MEDICINSKI GLASNIK - Aktuelno Ljekarska komora ZE - DO kantona

MEDICINSKI GLASNIK 

Official publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina 

Volume 8 Number 1, February 2011. 

ISSN 1840-0132 

Thematic issue: 

Medical biochemistry in clinical practice

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Medicinski Glasnik 

Official Publication of the Medical Association of 

Zenica-Doboj Canton 

Bosnia and Herzegovina 

Editorial Board 

Editor-iN-chiEf 

Selma Uzunović-Kamberović 

Zenica, Bosnia and Herzegovina 

MaNaGiNG 

Harun Drljević 

Zenica, Bosnia and Herzegovina 

Editors 

Adem Balić, Tuzla, Bosnia and Herzegovina 

Dubravka Bartolek, Zagreb, Croatia 

Branka Bedenić, Zagreb, Croatia 

Asja Čelebić, Zagreb, Croatia 

Josip Čulig, Zagreb, Croatia 

Filip Čulo, Mostar, Bosnia and Herzegovina 

Jordan Dimanovski, Zagreb, Croatia 

Branko Dmitrović, Osijek, Croatia 

Davorin Đanić, Slavonski Brod, Croatia 

Lejla Ibrahimagić-Šeper, Zenica, Bosnia and Herzegovina 

Tatjana Ille, Belgrade, Serbia 

Vjekoslav Jerolimov, Zagreb, Croatia 

Mirko Šamija, Zagreb, Croatia 

Ines Drenjančević-Perić, Osijek, Croatia 

Sven Kurbel, Osijek, Croatia 

Snježana Pejičić, Banja Luka, Bosnia and Herzegovina 

Belma Pojskić, Zenica, Bosnia and Herzegovina 

Asja Prohić, Sarajevo, Bosnia and Herzegovina 

Velimir Profozić, Zagreb, Croatia 

Radivoje Radić, Osijek, Croatia 

Amira Redžić, Sarajevo, Bosnia and Herzegovina 

Suad Sivić, Zenica, Bosnia and Herzegovina 

Sonja Smole-Možina, Ljubljana, Slovenia 

Vladimir Šimunović, Mostar, Bosnia and Herzegovina 

Adrijana Vince, Zagreb, Croatia 

Jasmina Vraneš, Zagreb, Croatia 

Živojin Žagar, Zagreb, Croatia 

Secretary: Tatjana Žilo; 

Proofreaders: Aras Borić (Bosnian, Croatian, Serbian), 

Glorija Alić (English), 

Cover: Senka Hukić “Star”

MEDICINSKI GLASNIK 

Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina 

Volume 8, Number 1, August 2011 

Free full-text online at: www.ljkzedo.com.ba, and www.doaj.org (DOAJ, Directory of Open Access 

Journals) 

Tematic issue: Medical biochemistry in clinical practice 

Content 

Review 1 Fetalna ehokardiografija u XV nedjelji trudnoće 

Fetal echocardiography in the 15 th week of gestation 

Adem Balić, Devleta Balić 

Original 

article 

9 Better stability of acenocoumarol compared to warfarin treatment in one-year observational, 

clinical study in patients with nonvalvular atrial fibrillation 

Aida Kulo, Jasna Kusturica, Elvedina Kapić, Fahir Bečić, Maida Rakanović-Todić, Lejla 

Burnazović-Ristić, Amila Mehmedović, Orhan Lepara, Nedžad Mulabegović 

15 Elastic characteristics of aorta in patients with epilepsy 

Namık Ozmen, Mehmet Senol Guney, Erdem Togrul, Omer Yiginer, Mustafa Aparci, 

Ejder Kardesoglu, Yılmaz Cingozbay, Mehmet Saracoglu, Bekir Sitki Cebeci 

19 Predictive value of fetal nuchal translucency 

Dragan Lončar 

24 Uticaj biometeoroloških faza na incidencu suicida 

Influence of biometeorological phases on incidence of suicides 

Vladimir Gajić, Dragan Milojević, Jasminka Smailagić, Nela Đonović, Suzana Matejić, 

Sanja Gajić 

31 Colorectal cancer early detection program integrated in practice of family physicians 

Sanda Pribić, Ljiljana Trtica - Majnarić, Rudika Gmajnić, Marko Lukić, Nada Prlić 

39 Physicians overestimate patient’s knowledge of the process of informed consent: a 

cross-sectional study 

Marko Jukic, Slavica Kozina, Goran Kardum, Rosemary Hogg, Slavica Kvolik 

46 Antibakterijski učinak materijala za punjenje korijenskih kanala, podloge i ispune 

kaviteta 

Antibacterial effect of the materials for the root canal filling, bases and material for 

the cavity fillings 

Suzana Ferk, Paris Simeon, Goranka Prpić Mehičić, Smilja Kalenić, Ivica Anić, Silvana 

Jukić 

Notes 53 Utjecaj trajanja dijabetesa i neregulirane glikemije na nastanak retinopatije 

Influence of lasting diabetes and non-adjustable glycosylated haemoglobin on occurrence 

of retinopathy 

Salem Alajbegović, Azra Alajbegović, Jasmina Omerović, Adnan Mušanović, Elmedin 

Lačić 

56 Epidemiološke karakteristike multiple skleroze u Bosni i Hercegovini 

Epidemiology of multiple sclerosis in Bosnia and Herzegovina 

Azra Alajbegović, Salem Alajbegović, Jasminka Đelilović-Vranić, 

60 Anxiety, splint treatment and clinical characteristics of patients with osteoarthritis of 

temporomandibular joint and dental students – a pilot study 

Tomislav Badel, Sandra Kocijan Lovko, Dijana Podoreški, Ivana Savić Pavčin, Josipa 

Kern 

Case 

reports 

63 Hronični epiduralni hematom dijagnosticiran kao meningeom 

Chronic epidural haematoma mimicking meningioma 

Hakija Bečulić, Rasim Skomorac, Aldin Jusić, Alma Mekić-Abazović, Alma Bajtarević

66 Placenta previa percreta with bladder invasion 

Siniša Šijanović, Mirjana Rubin, Zlatko Topolovec, Domagoj Vidosavljević, Ivanka 

Šijanović 

68 Endometrijalni karcinom povezan s porastom CA 15_3 i CA 125 u bolesnice s karcinomom 

dojke liječene tamoksifenom 

Endometrial cancer associated with an increase of CA 15_3 and CA 125 in tamoxifen 

treated patients with breast cancer 

Alma Mekić-Abazović, Hakija Bečulić, Miralem Musić, Almir Fajkić, Senad Dervišević 

71 Primary malignant melanoma of gallbladder 

Vlatka Pitlović, Ferid Latić, Hrvoje Pitlović, Mario Rupčić, Darko Jurišić, Azra Latić 

Tematic issue: Medical biochemistry in clinical practice 

Editorial 75 Medical biochemistry in everiday clinical praxis 

Original 

article 

76 Association of PPARG and LPIN1 gene polymorphisms with metabolic syndrome 

and type 2 diabetes 

Povezanost polimorfizama PPARG i LPIN1 gena s metaboličkim sindromom i dijabetesom 

tipa 2 

Tamer Bego, Tanja Dujic, Barbara Mlinar, Sabina Semiz, Maja Malenica, Besim Prnjavorac, 

Barbara Ostanek, Janja Marc, Adlija Čaušević 

84 Analysis of CYP3A4*1B and CYP3A5*3 polymorphisms in population of Bosnia and 

Herzegovina 

Analiza CYP3A4*1B i CYP3A5*3 polimorfizama u populaciji iz Bosne i Hercegovine 

Sabina Semiz, Tanja Dujić, Barbara Ostanek, Besim Prnjavorac, Tamer Bego, Maja 

Malenica, Barbara Mlinar, Janja Marc, and Adlija Čaušević 

90 Optimisation of methods for quantifying plasma mRNA levels from genes responsible 

for coronary artery plaque development and destabilization 

Darko Černe, Irma Štern, Igor Kranjec, Janja Marc 

97 Cystatin C in sera of patients with aggressive non-Hodgkin B-cell lymphoma 

Cistatin C u serumu pacijenata s agresivnim ne-Hodgkinovim limfomom B ćelija 

Adaleta Softić, Lejla Begić, Alma Halilbašić, Janko Kos 

101 Local CD4+, CD8+ and CD56+ reactions to lung cancer in regard to pathohistological 

type and clinical stage 

Lokalna CD4+, CD8+ i CD56+ reakcija na karcinom pluća u zavisnosti od 

patohistološkog tipa i kliničkog stadija oboljenja 

Edin Jusufovic, Ermina Iljazovic, Mitja Kosnik, Dragan Keser, Peter Korosec, Edin Zukic, 

Besim Prnjavorac, Rifat Sejdinović, Ekrem Ajanović 

109 Interleukin 18 expression in the primary breast cancer tumour tissue 

Ekspresija interleukina 18 u tkivu primarnog tumora raka dojke 

Nahida Srabović, Zlata Mujagić, Jasminka Mujanović-Mustedanagić, Zdeno Muminović, 

Elmir Čičkušić 

116 Use of amino-terminal pro-B type natriuretic peptide as the parameter for long term 

monitoring of water overload in patient with chronic kidney diseases 

Analiza amino-terminalnog pro-B-tipa natriuretskog peptida kao parametra retencije 

tekućine u praćenju bolesnika s hroničnom bubrežnom bolesti 

Besim Prnjavorac, Kadrija Abduzaimović, Jasna Jukić, Rifat Sejdinović, Ekrema Mujarić, 

Nedžada Irejiz, Almira Hadžović-Džuvo, Radivoj Jadrić, Adlija Čaušević, Sabina Semiz, 

Tamer Bego, Maja Malenica 

121 B-type natriuretic peptide (BNP) serum levels in rats after forced repeated swimming 

stress 

Koncentracija B-tipa natriuretskog peptida (BNP) u serumu štakora nakon forsiranog, 

ponavljanog stresa, uzrokovanog plivanjem 

Almira Hadžovic-Džuvo, Amina Valjevac, Nesina Avdagić, Orhan Lepara, Asija 

Zaćiragić, Radivoj Jadrić, Jasmin Alajbegović, Besim Prnjavorac

126 Association of homocysteine with traditional and non-traditional risk factors in 

patients with atherosclerotic vascular disease 

Povezanost homocisteina s tradicionalnim i netradicionalnim faktorima rizika kod 

pacijenata s aterosklerotskom vaskularnom bolesti 

Emina Kiseljaković, Amina Valjevac, Sabaheta Hasić, Emina Nakaš-Ićindić, Alen 

Džubur, Radivoj Jadrić 

134 Coagulation factor VIII activity in diabetic patients 

Aktivnost VIII faktora koagulacije u pacijenata s diabetes mellitusom 

Nermina Babić, Amela Dervišević, Jasminko Huskić, Miralem Musić 

140 Time-dependent responses of rat troponin I and cardiac injury following isoproterenol 

administration 

Vremenski ovisni odgovori troponina I štakora i oštećenja miokarda nakon administracije 

izoproterenola 

Sabaheta Hasić, Radivoj Jadrić, Emina Kiseljaković, Amina Valjevac, Zakira 

Mornjaković, Mira Winterhalter-Jadrić 

146 Blood iron stores reduction affects lipoprotein status - a potential benefit of blood donation 

Smanjivanje zaliha željeza u krvi utiče na status lipoproteina – potencijalna korist 

od darivanja krvi 

Radivoj Jadrić, Sabaheta Hasić, Emina Kiseljaković, Jozo Ćorić, Besim Prnjavorac, Mira 

Winterhalter-Jadrić 

151 Hematologic and laboratory parameters in patientis with peptic ulcer bleeding 

treated by two modalities of endoscopic haemostasis and proton pompe inhibitors 

Praćenje hematoloških i laboratorijskih parametara kod krvarećeg ulkusa 

primjenom dva protokola hemostaze, endoskopijom i inhibitorima protonske pumpe 

Amila Mehmedović-Redžepović, Rusmir Mesihović, Besim Prnjavorac, Aida Kulo, 

Kalajdžija Merlina 

158 Evaluation and treatment of cardiovascular diseases in patients on hemodialysis – 

single center experience 

Analiza dijagnostike i liječenja kod kardiovaskularnog komorbiditeta dijaliznih 

pacijenata – iskustvo jednog centra 

Halima Resić, Besim Prnjavorac, Fahrudin Mašnić, Selma Ajanović, Nihad Kukavica, 

Amela Bećiragić 

163 Adenosine deaminase and C-reactive protein in diagnosing and monitoring of rheumatoid 

arthritis 

Adenozin deaminaza i C-reaktivni protein u dijagnostici i praćenju reumatoidnog 

artritisa 

Milada Nalesnik, Jasminka Mehanović Nikolić, Slavica Jandrić 

Medicinski Glasnik is indexed by MEDLINE, Science Citation Index Expanded (SciSearch ® ), and 

Journal Citation Reports/Science Edition, EMBASE (Exerpta Medica), Scopus, EBSCO.

REVIEW 

Fetalna ehokardiografija u XV nedjelji trudnoće 

Adem Balić 1 , Devleta Balić 2 

1 Služba za ginekologiju i perinatologiju, Dom zdravlja u Tuzli, 2 Zavod za humanu reprodukciju „Dr. Balić” Tuzla 

Corresponding author: 

Adem Balić, 

Služba za ginekologiju i perinatologiju, 

Dom zdravlja u Tuzli 

Kojšino 25, 75000 Tuzla 

Phone: +387 35 286 724; 

fax.: +387 35 286-724; 

E-mail: badem@bih.net.ba 

Originalna prijava: 

18. februar 2010.; 

Korigirana verzija: 

04. mart 2010.; 

Prihvaćeno: 

13. april 2010. 

SAŽETAK 

Med Glas Ljek komore Zenicko-doboj kantona 2011; 8(1):1-8 

Brzi razvoj ultrazvučne tehnike omogućio je postizanje vrlo visoke 

rezolucije slike otvorivši time put ka kvalitetnijem prikazu svih fetalnih 

struktura, pa i fetalnog srca. Uobičajeno je da se ultrazvučni 

pregled srca obavlja transabdominalnim putem, iza XX nedjelje, 

kada je sa sondama frekvence 3-5 MHz moguće dobiti kvalitetan 

prikaz svih relevantnih srčanih struktura. S obzirom da se formiranje 

fetalnog srca najvećim dijelom završava do kraja XIV nedjelje, 

logično je da se nametnula ideja o vaginalnom ultrazvučnom pregledu 

u XV nedjelji sa sondama jačine 5-9 MHz. U tom periodu, 

dužina ploda kreće se između 9 i 10 cm što daje pretpostavku da 

se vaginalnom sondom može izvršiti kvalitetan pregled i fetalnog 

srca. U ovom preglednom članku prikazani su svi standardni presjeci 

fetalnog srca (4 komore, izlazni trunkusi, presjek kroz tri velike 

krvne žile, te longitudinalni prikaz aortalnog i duktalnog luka) 

vaginalnim pregledom u XV nedjelji i abdominalnim u XX i XXII 

nedjelji. Transvaginalni pregled standardnih presjeka fetalnog srca 

moguć je i u XV nedjelji, te bi ga trebalo uraditi kod svih trudnica 

sa opterećenom ličnom i reproduktivnom anamnezom, kao i 

kod onih sa pozitivnim ultrazvučnim markerima na hromozomske 

aberacije. 

Ključne riječi: ehokardiografija, fetus, XV nedjelja trudnoće 

1

2 

Medicinski Glasnik, Volumen 8, Number 1, February 2011 

UVOD 

Urođene srčane mane su najčešće fetalne anomalije, 

a njihova incidencija se kreće od 3-10 na 

1.000 rođenih (1-3). One mogu biti izolovane, 

ali i udružene, kako s drugim anomalijama, tako 

i s genetskim greškama (4). Učestalost srčanih 

mana je 6,5 puta veća nego učestalost hromozomskih 

aberacija i četiri puta veća nego defekata 

neuralne cijevi (5). Srčane mane se pojavljuju 

kod 4% živorođene djece; 20% ih umire u 

prvom mjesecu, 50% u prvoj godini, dok ostali 

imaju različite probleme koji značajno utječu na 

rast i razvoj (3,6). 

U dječijoj kardiohirurgiji došlo je do značajnog 

napretka u rješavanju velikog broja urođenih 

srčanih mana. Najvećim napretkom smatra se 

korekcija transpozicije velikih arterija kojom se 

zamijene pozicije aorte i plućne arterije još u neonatalnom 

periodu, te tako uspostavi normalna 

cirkulacija vrlo brzo nakon rođenja (7). 

Specifičnost fetalne cirkulacije jeste osnovni razlog 

zašto se većina srčanih mana ne manifestuje 

u trudnoći. Četiri su glavne karakteristike fetalne 

cirkulacije: povišena plućna vaskularna rezistencija 

i smanjen protok kroz pluća, niska placentalna, 

odnosno sistemska vaskularna rezistencija, 

postojanje komunikacija između sistemske i 

plućne cirkulacije i paralelna dispozicija obje komore 

koje simultano pumpaju krv u istu, sistemsku 

cirkulaciju. Zbog ovih karakteristika fetalne 

cirkulacije, pojedine srčane mane se dobro podnose 

in utero, a srčanu insuficijenciju ispoljavaju 

tek u prvim nedjeljama života (1,6). 

Uvođenje fetalne ehokardiografije (8) omogućilo 

je postavljanje tačne dijagnoze još u trudnoći, a 

time i daljni adekvatan nadzor, te porođaj i operativni 

zahvat u odgovarajućem kardiološkom 

centru. U većini evropskih zemalja preporučeni 

period za pregled fetalnog srca jeste XVIII i 

XXV nedjelja (2,9,10,11). Vodeći stručnjaci za 

fetalnu ehokardiografiju smatraju da je optimalna 

veličina trudnoće za pregled fetalnog srca između 

XVIII i XX nedjelje, transabdominalnom sondom 

5-7,5 MHz (10), mada Yagel (1,10) predlaže 

da se fetalna ehokardiografija radi na početku II 

trimestra (14-16 nedjelja) transvaginalno, a da 

se ponovi između XXII i XXIII nedjelje. I drugi 

su autori ukazali na mogućnost i značaj pregleda 

embrionalnog srca na kraju I trimestra trudnoće 

(12-15), čime je trudnicama omogućen izbor i 

za prekidom trudnoće još u prvom trimestru ako 

se radi o teškoj srčanoj mani (3,10,16), ili se dobije 

dragocjeno vrijeme za druge dijagnostičke 

pretrage i konačnu dijagnozu (17,18). Ukoliko 

se radi o operabilnoj srčanoj mani, kontaktira se 

referentni kardiohirurški centar pod čijim nadzorom 

će biti daljnji tok trudnoće, te vrijeme i mjesto 

poroda. Na taj način značajno se povećavaju 

šanse za uspjeh nakon postnatalnog operativnog 

tretmana (19). U nekim slučajevima, kao što je to 

kod smetnji srčanog ritma, moguće je liječenje i 

u vrijeme trudnoće (20). 

PRENATALNA DIJAGNOZA SRČANIH MANA 

Fetalni skrining u XIII ili XIV nedjelji ukazuje na 

hromozomske aberacije, ali indirektno i na srčane 

mane, te bi pozitivan nalaz trebao biti indikacija 

za fetalnu ehokardiografiju (4, 21-25), kao i trudnice 

s dijabetesom (26) ili pozitivnom ličnom, 

porodičnom ili reproduktivnom anamnezom u 

vezi sa srčanim oboljenjima u ranijim trudnoćama 

ili kod bliže rodbine (27-29). 

Brzi razvoj ultrazvučne tehnike kao što je obojeni 

dopler, pulsni dopler, power dopler, 3D, 4D, 

B-tok (B-flow), te usavršavanje digitalne tehnike, 

omogućili su dobijanje vrlo visoke rezolucije 

slike, a time i pregled fetalnog srca i u manjoj 

trudnoći (11,12,15). 

S obzirom da se formiranje fetalnog srca najvećim 

dijelom završava do kraja XIV nedjelje, 

logično je da se pojavila ideja o vaginalnom 

pregledu sa sondom visoke rezolucije već u XV 

nedjelji. U tom periodu dužina ploda (CRL) se 

kreće između 9 i 10 cm što omogućava da se vaginalnom 

sondom izvrši kvalitetan pregled jer je 

plod još uvijek u njenom ‘’dometu’’ (10-12). S 

druge strane, na ovaj se način postiže visoka rezolucija 

slike jer je između ploda i fetalnog srca 

samo tanki zid uterusa, te je moguće napraviti 

sve referentne presjeke potrebne za pregled srca 

(28, 29). Uz to, kod ovoga pregleda položaj ploda, 

debljina pacijentice, te smještaj posteljice, ne 

utječu na kvalitet pregleda. 

TEHNIKA ULTRAZVUČNOG PREGLEDA FETALNOG 

SRCA 

Prenatalna dijagnostika srčanih mana temelji se 

prije svega na ocjeni prikaza ‘’četiri komore’’ 

koji se uobičajeno izvodi između XVIII i XXV

nedjelje trudnoće, a koji je u većini razvijenih 

država dio prenatalnog skrininga (2,11,12). Prije 

nego se pristupi traženju presjeka četiri komore 

neophodna je orijentacija u vidu prikaza gornjeg 

abdomena koji omogućuje prikaz lokacije želuca, 

jetre, aorte, vene kave inferior u odnosu na 

kičmu, a time i položaja unutrašnjih organa (28) 

(Slika 1). Poslije toga se traži presjek kroz četiri 

šupljine koji predstavlja osnovu za procjenu 

normalne srčane anatomije (11,28,29) (Slika 2). 

Kada se nađe traženi presjek, slika se uveća za tri 

do četiri puta, pa se tek onda analizira. Prvo se 

procijeni veličina srca u odnosu na grudni koš, 

a koja ne bi trebala biti veća od jedne trećine. 

Položaj srca je normalan kada je vrh okrenut 

prema lijevo, tako da ugao, kojeg čine interventrikularni 

septum i zamišljena linija od sternuma 

do kičme, iznosi od 35° do 45°, i nalazi se iznad 

želuca (16,28,29). Daljnjom analizom segmentne 

građe srca isključuje se postojanje perikardijalnog 

izljeva, te izgled i veličina komora koje su 

približno jednake. Desna komora se raspoznaje 

po položaju (bliža je zidu grudnog koša), niže 

Balić et al Fetalna ehokardiografija 

Slika 1. Presjek na nivou gornjeg abdomena: XX nedjelja (lijevo) i XV nedjelja (desno) (Balić A., 2007, 2009.) 

usađenom trikuspidalnom valvulom u odnosu 

na mitralnu, kao i nešto grubljim unutrašnjim 

zidovima zbog postojećih trabekula i Morganijevog 

mišića. Interventrikularni septum ima 

zadebljanu bazu na apikalnom kraju i istanjeni 

membranozni dio ka atrioventrikularnom spoju 

na koji se jednim dijelom pripajaju valvule (30). 

Pretkomore su manje, ovalnije, s međuatrijskim 

septumom u kojem se raspoznaje foramen ovale 

i njegova valvula koja treperi unutar lijevog 

atrija. Valvule se inače posmatraju korištenjem 

funkcije ‘’cine loop’’ koju imaju svi noviji aparati, 

a koja omogućuje vraćanje dvadesetak i više 

sličica. Drugim riječima, možemo vratiti i usporiti 

otvaranje i zatvaranje valvula. Detaljna ocjena 

četverokomorskog prikaza po ovim kriterijima 

može otkriti većinu srčanih mana (2,10,31). 

Ako se tome doda i mogućnost prikaza izlaznih 

komorskih trunkusa, učestalost otkrivanja srčane 

mane penje se i do 85% (2)! 

Postepenom rotacijom sonde iz transverzalne u 

uzdužnu ravan grudnog koša, a uz praćenje duže 

ose lijeve komore, dobije se izlazni trakt u smjeru 

Slika 2. Četverokomorski prikaz srca: u XX nedjelji (lijevo) i u XV nedjelji (desno) (Balić A., 2010, 2009.) 

3

4 


Slika 3. Prikaz lijevog izlaznog trunkusa: u XX nedjelji (lijevo) i u XV nedjelji (desno) (Balić A., 2010, 2009.) 

prema desnom ramenu ploda (28,29,32,33) (Slika 

3). Prednji zid se nastavlja na interventrikularni 

septum, a zadnji produžava u prednji zalistak 

mitralne valvule, uz istovremenu vizualizaciju 

cijele valvule. Blagom rotacijom sonde prema 

vani, a u istoj ravni, dobije se i presjek kroz dužu 

osu desne komore na kojem se može raspoznati 

arterija pulmonalis kako izlazi iz desne komore 

u smjeru prema nazad i lijevo, u svom karakterističnom 

odnosu s lukom aorte (28,29,32,33) 

(Slika 4). Pomenute velike arterije križaju se pod 

pravim uglom, a eventualni izostanak tog križanja 

ukazuje na transpoziciju velikih krvnih žila. 

PRESJEK KROZ KRAĆU OSU VELIKIH KRVNIH 

ŽILA 

Dva izlazna trunkusa mogu biti prikazana simultano 

u jednoj kosoj parasagitalnoj ravni čime se 

omogućava i pregled perimembranoznog dijela 

interventrikularnog septuma (28,29,32,33). 

Prikazom izlaznih trunkusa može se ustanoviti 

postojanje dva odvojena arterijska zaliska, da su 

izlazni trunkusi približno jednake širine, kao i da 

su postavljeni pod pravim uglom. 

Desni izlazni trunkus nastavlja se u plućnu arteriju 

koja je usmjerena ravno i nazad prema kičmi. Plućna 

je arterija veoma kratka; brzo se dijeli na tri grane: 

desnu i lijevu plućnu arteriju i ductus arteriosus 

(koji je vizualno produžetak glavne arterije). 

Arterija koja proizlazi iz lijevog trunkusa je aorta. 

Za razliku od plućne arterije, ascendentna aorta 

je relativno duga, s tim da prve veće grane idu 

prema glavi. 

Prikaz tri krvne žile 

Prikaz tri krvne žile dobiva se na jednoj kosoj poprečnoj 

ravni visoko u grudnom košu (12, 28, 29, 

32, 33). S obzirom da se aortni luk nalazi više u 

prsima nego ‘’duktalni luk’’ koji leži s njegove 

lijeve strane, zamišljena ravan mora biti nagnuta 

prema plodovoj lijevoj strani da bi se prikazala 

oba luka. 

Cilj ovoga presjeka jeste prikazati tri velike krvne 

žile (pulmonarnu arteriju, aortu i venu cava 

superior) pri čemu je pulmonarna arterija smještena 

više prema naprijed (Slika 5). 

Aortalni luk i pulmonarna arterija, oko 20 sedmice, 

približno su jednaki širinom. U većoj trudnoći, pul- 

Slika 4. Prikaz desnog izlaznog trunkusa: u XX nedjelji (lijevo) i u XV nedjelji (desno) (Balić A., 2009, 2010.)

Slika 5. Prikaz tri velike krvne žile: u XX nedjelji (lijevo) i u XV nedjelji (desno) (Balić A., 2009.) 

monarna arterija je nešto veća nego aorta, a izraženija 

razlika može ukazivati na koarktaciju aorte. 

Longitudinalni prikaz aortnog luka 

Jednim kosim, sagitalnim presjekom može se 

prikazati lijevi izlazni trunkus, luk aorte i silazna 

aorta (28,29). Aortni luk se proteže od prednje 

desne strane ploda prema straga i lijevo. Stoga 

je ova ravan najbolje locirana ukoliko se sondom 

prelazi od srednjeg sagitalnog dijela kičme fetusa 

i ukoliko je sonda usmjerena na lijevi dio kičme i 

ukošena lagano prema desnoj strani ploda. 

Normalno je da aortni luk formira uski ‘’pastirski 

štap’’; širi aortni luk je karakteristika premještanja 

velikih arterija (Slika 6), a uži može ukazivati 

i na koarktaciju aorte. 

Aortni luk daje grane za glavu i vrat. Prikaz aortnog 

luka bez ogranaka za glavu i vrat može biti 

posljedica srčane mane ili nepravilnog prikaza 

ravni koji pokazuje tzv. ‘’kanalni luk’’. U ostalim 

presjecima, plućna arterija, arterijski kanal i silazna 

aorte vide se u kontinuitetu. 

M-MOD ZAPIS 


M-mod predstavlja osnovu za detaljnu analizu 

kretnji određenih intrakardijalnih struktura i njihovu 

uzročno-posljedičnu vremensku povezanost 

u toku srčanog ciklusa (20). Pravu vrijednost 

M-mod ima tek u kombinaciji s dvodimenzionalnom 

‘’živom’’ slikom koja omogućuje pravilno 

postavljanje kursora u željenu ravan. M-mod kursor 

mora da prođe kroz određene srčane strukture, 

a da bi se to postiglo obično se koristi dvodimenzionalna 

slika u ravni četiri srčane šupljine 

(Slika 7). 

OBOJENI I PULSNI DOPLER U PREGLEDU FETAL- 

NOG SRCA 

Obojeni ili kolor dopler predstavlja drugu fazu 

pregleda fetalnog srca, a koji značajno obogaćuje 

informacije koje nudi siva skala (17,34). Njegov 

je najveći značaj u jednostavnom i brzom prikazivanju 

homogenog protoka kroz srce i velike 

krvne žile, te njihovo križanje pri izlasku iz srca. 

Osim toga, kolor dopler olakšava i vizualizaciju 

Slika 6. Longitudinalni prikaz aortnog luka: u XX nedjelji (lijevo) i u XV nedjelji (desno) (Balić A., 2010, 2009.) 

5

6 


Slika 7. M-mod zapis u XV nedjelji (Balić A., 2009.) 

defekta interventrikularnog septuma, te valvularnu 

regurgitaciju. Patološki protok ima karakterističnu 

žućkasto-zelenkastu boju (Slika 8), a 

pulsnim se doplerom, u tom slučaju, registruje 

reverzni protok (Slika 9). 

STIC 

STIC (engl. spatio-temporal image correlation) 

predstavlja novi način kliničkog ispitivanja građe 

i funkcije fetalnog srca (33-41). Ta novina nudi 

jednostavno korištenje 4D ultrazvučne tehnike za 

prikupljanje velikog broja podataka vezanih za 

građu fetalnog srca, i u vremenu i u prostoru. Cijeli 

se postupak sastoji iz dva dijela. Prvi je skeniranje 

nekoliko srčanih ciklusa u trajanju od 12 do 

15 sekundi. Drugi je analiza dobivenih podataka 

putem kompjuterskog programa koji omogućuje 

analizu svakog dijela srca u prostoru kada je ono 

zaustavljeno u nekoj fazi srčanog ciklusa. Takođe 

se analizira i u ‘’vremenu’’, tj. kada srce radi, 

s tim da se može značajno usporiti što olakšava 

Slika 8. Kolor dopler prikaz interventrikularnog septalnog 

defekta u XV nedjelji (Balić A., 2009.) 

vizualizaciju svih relevantnih srčanih struktura, 

a time i prepoznavanaje velikog broja srčanih 

mana. 

Ovaj program značajno olakšava vrlo kompleksan 

pregled fetalnog srca, te otvara mogućnost 

dijagnostikovanja i vrlo kompleksnih urođenih 

srčanih mana, čak i u XV nedjelji trudnoće (14, 

15). 

U zaključku, u standardnoj fetalnoj ehokardiografiji 

segmentna građa srca ispituje se različitim 

presjecima kojima se vizualizira poprečni presjek 

gornjeg abdomena, četiri šupljine, izlazni trunkusi, 

tri velika krvna suda i longitudinalni prikaz 

luka aorte. 

Na ovaj način mogu se otkriti ventrikularni i atriventrikularni 

septalni defekti, koarktacija aorte, 

stenoza a. pulmonalis, stenoza aorte, tetralogija 

Fallot, sindrom hipoplastičnog lijevog srca, transpozicija 

velikih krvnih žila, te kardiomegalija, 

perikardijalni izljev, patološka pozicija srca, poremećaji 

ritma i frekvence srčanog rada. 

Zahvaljujući usavršavanju ultrazvučnih uređaja 

danas je moguće sve ove presjeke prikazati i vaginalnom 

sondom u XV nedjelji, kada je srce potpuno 

formirano, te ne treba čekati XX nedjelju. 

Pregled je složen i dug, ali je moguć i to na istoj 

opremi na kojoj se inače radi klasična fetalna 

ehokardiografija u XX nedjelji. Jedini problem 

jeste što se pregled mora uraditi u XV nedjelji jer 

u manjoj trudnoći to nije moguće zbog nedovoljne 

razvijenosti i veličine srca, a u većoj trudnoći 

fetus, zbog svoje veličine, može izaći izvan ‘’dometa’’ 

vaginalne sonde. 

To je osobito važno za trudnice koje spadaju u 

grupu visokog rizika za rađanje djece sa srčanom 

greškom, kod kojih bi se kvalitetan probir mogao 

izvršiti već u XV nedjelji, te se na taj način može 

Slika 9. Pulsni dopler trikuspidalne regurgitacije u XV nedjelji 

(Balić A., 2009.)

pomoći roditeljima da na vrijeme donesu odluku 

za prekid trudnoće, ako se ustanovi teška mana, 

ili o nastavku trudnoće i ponovnom pregledu između 

XX i XXIV nedjelje. 

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JA, Popp RL, Sahn DJ, Schiller NB, Tajik A, 

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IZJAVE 


Komercijalni ili potencijalni dvostruki interes ne 

postoji. 

14. Viñals F, Ascenzo R, Naveas R, Huggon I, Giuliano 

A. Fetal echocardiography at 11 + 0 to 13 + 6 weeks 

using four-dimensional spatiotemporal image correlation 

telemedicine via an Internet link: a pilot study. 

Ultrasound Obstet Gynecol 2008; 31:633-8. 

15. Turan S, Turan OM, Ty-Torredes K, Harman CR, 

Baschat AA. Standardization of the first-trimester 

fetal cardiac examination using spatiotemporal image 

correlation with tomographic ultrasound and colour 

Doppler imaging. Ultrasound Obstet Gynecol 

2009; 33:652-6. 

16. Bajić S. Dijagnostika najčešćih urođenih anomalija 

fetalnog srca. U: Zbornik radova II kongresa ginekologa 

i perinatologa u BiH, Sarajevo 1.-3.10.2009. 

Müller, Sarajevo, 2009: 19. 

17. Balić A. Antenatalna dijagnostika fetalnih anomalija 

i hromozomskih bolesti. U: Balić A, ur. Perinatologija. 

Tuzla: PrintCom, 2007:76-85. 

18. Balić D, Balić A. Antenatalna dijagnostika fetalnih 

anomalija i hromozomskih abnormalnosti. Pedijatrija 

danas 2008; 4:42-52. 

19. Chaoui R. Fetal echocardiography: state of the art of 

the state of the heart. Ultrasound Obstet Gynaecol 

2001; 17:277-84. 

20. Vesel S. Diagnostika in obravnava strkturnih srčnih 

napak in motenj srčnega ritma pri plodu: Diagnosis 

and Management of fetal Cardiac abnormalities. 

Medicinski razgledi 2005; 44 (supl 2):119-26. 

21. Hyett JA, Perdu M, Sharland G, Snijdeers RSM, Nikolaides 

KH. Using fetal translucency to screen for 

major congenitaal cardiac defect at 10-14 weeks of 

gestation: population based cohort study. Br J Obstet 

Gynaecol 1999; 18:81-5. 

22. Zosmer N, Souter VL, Chan CSY, Hugon IC, Nikolaides 

KH. Early diagnosis of major cardiac defects 

in chromosomally normal fetuses with increased nuchal 

translucency. Br J Obstet Gynaecol 1999; 106: 

829-33. 

23. Balić A, Balić D. The role of transvaginal 4D ultrasonography 

in detection chromosomal anomalities 

aat 13-14 weeks. U: Abstract book of the XVIII 

FIGO World congress of gynecology and obstetrics, 

Kuala Lumpur, Malaysia, 2006. FIGO, London, 

2006., Abstract V3.9.1, str. 136.. 

24. Balić A, Balić D. Skrining na hromozomske aberacije 

u XIII ili XIV nedjelji trudnoće. U: Zbornik 

radova simpozijuma Perinatalni trendovi u Bosni i 

Hercegovini. Sarajevo, ANUBIH 2006:135-8. 

25. Balić A, Balić D. Ultrazvučni pregled u XIV 

nedjelji.U: Zbornik radova Perinatalnih dana BiH. 

Tuzla, Printcom 2008: 69-74. 

26. Jaeggi ET, Fouron JC, Bland JM, Tekay A, Thilaganathan 

B. Fetal cardiac performance in uncomplicated 

and well-controlled maternal type I diabetes. 

Ultrasound Obstet Gynaecol 2001; 17:311-5 

27. Sklansky MS. Prenatal screening for congenital heart 

disease: a moving proposal. J Ultrasound med 

2007; 26:1-7. 

7

8 


28. Huggon IC. Practical guide to fetal echocardiography. 

Prenat Neonat Med 2001; 6:38-53. 

29. Yagel S, Cohen SM, Achiron R. Examination of fetal 

heart by five short-axis view: a proposed screening 

method for comprehensive cardiac evaluation. Ultrasound 

Obstet Gynaecol 2001; 17:367-9. 

30. Ibrahimović J, Begić H. Ventrikularni septalni defekt 

–rezultati desetogodišnjeg praćenja. Pedijatrija 

danas 2007; 3:194-200. 

31. Jouannic JM, Gavard L, Fermont L, Le Bidois J, Parat 

S, Vouhé PR, Dumez Y, Sidi D, Bonnet D. Sensitivity 

and specificity of prenatal features of physiological 

shunts to predict neonatal clinical status in 

transposition of the great arteries. Circulation 2004; 

110:1743-6. 

32. Yagel S, Arbel R, Anteby EY, Raveh D, Achiron R. 

The three vessels and trachea view (3VT) in fetal 

cardiac scanning. Ultrasound Obstet Gynecol 2002; 

20:340-5. 

33. DeVore GR, Polanco B, Sklansky MS, Platt LD. The 

‘stic’ technique: a new method for examination of 

the fetal outflow tracts using three-dimensional ultrasound. 

Ultrasound Obstet Gynecol 2004; 24:72- 

82. 

34. Turan S, Turan OM, Maisel P, Gaskin P, Harman 

CR, Baschat AA. Three-dimensional sonography in 

the prenatal diagnosis of aortic arch abnormalities. J 

Clin Ultrasound 2009; 37:253-7. 

35. Hartung J, Kalache KD, Chaoui R. Three-dimensional 

power Doppler ultrasonography (3D-PDU) in 

fetal diagnosis. Ultraschall Med 2004; 25:200-5. 

Fetal echocardiography in the 15 th week of gestation 

36. DeVore GR, Falkensammer P, Sklansky MS, Platt 

LD. Spatiotemporal image correlation (STIC): new 

technology for evaluation of the fetal heart. Ultrasound 

Obstet Gynecol 2003; 22:380-7. 

37. Viñals F, Poblete P, Giuliano A. Spatio-temporal 

image correlation (STIC): a new tool for the prenatal 

screening of congenital heart defects. Ultrasound 


38. Volpe P, Campobasso G, Stanziano A, De Robertis 

V, Di Paolo S, Caruso G, Volpe N, Gentile M. Novel 

application of 4D sonography with B-flow imaging 

and spatio-temporal image correlation (STIC) in the 

assessment of the anatomy of pulmonary arteries in 

fetuses with pulmonary atresia and ventricular septal 

defect. Ultrasound Obstet Gynecol 2006; 28:40-6. 

39. Turan S, Turan A, Baschat A. Three- and four-dimensional 

fetal echocardiography. Fetal Diagn Ther 

2009; 25:361–72. 

40. Stoeckl C. An automated approach to visualize 

standard views of the fetal heart. New findings, 

new visualization planes. GE Healthcare Zipf, Austria 

[Online] (2009). http://www.volusonclub.net/ 

whitepapers&id=345 (3. februar 2010.) 

41. Falkensammer P. Spatio-temporal Image correlation 

for volume ultrasound studies of the fetal heart. 

GE Healthcare, Zipf, Austria [Online] 2010. http:// 

www.volusonclub.net/whitepapers&id=181 (3. februar 

2010). 

Adem Balić 1 , Devleta Balić 2 

Department of Obstetric and Gynaecology, Health Centre Tuzla, Human reproduction centre “Dr Balić” Tuzla 

ABSTRACT 

A fast development of the ultrasound technology has enabled a very high resolution of images thus opening 

a possibility for better quality of images of all fetal structures including fetal heart. Transabdominal 

ultrasound examination of heart is commonly conducted after the 10th week when it is possible to get 

a quality image of all relevant heart structures using 3-5MHz probes. Having in mind that fetal heart 

is mostly formed by the end of the 14th week, an idea of a vaginal ultrasound examination in the 15th 

week using 5-9MHz probes has been a logical one. In that period the length of the fetus ranges from 9 

to 10 centimeters, which is a basis for an assumption that a quality examination of the fatal heart may 

be conducted using the vaginal probe. This paper is showing all standard cross-sections of fetal heart (4 

chambers, outflow tracts, cross-section of three great vessels and longitudinal view of aortic and ductal 

arches) obtained by vaginal examination in the 15th week and abdominal examination in the 20th and 

22nd week. 

The transvaginal examination of fetal heart is possible in the 15th week and it is recommended to be 

done in all pregnant women with problematic personal and reproductive anamnesis and those with positive 

ultrasonographic markers for chromosomal defects. 

Key words: echocardiography, fetus, 15 weeks 

Originalna prijava: 18. februar 2010.; Korigirana verzija: 04. mart 2010.; Prihvaćeno: 13. april 2010.

ORIGINAL ARTICLE 

Better stability of acenocoumarol compared to warfarin treatment 

in one-year observational, clinical study in patients with 

nonvalvular atrial fibrillation 

Aida Kulo 1 

, Jasna Kusturica 1 

, Elvedina Kapić 1 

, Fahir Bečić 1 

, Maida Rakanović-Todić 1 

, Lejla Burnazović- 

Ristić 1 

, Amila Mehmedović 2 

, Orhan Lepara 3 

, Nedžad Mulabegović 1 

1 

Institute of Pharmacology, Clinical Pharmacology and Toxicology, School of Medicine, University of Sarajevo, 2 

Clinic for Gastroenterohepathology, 

Clinical Centre of the University of Sarajevo, 3 

Institute of Physiology and Biochemistry, School of Medicine, University of 

Sarajevo; Sarajevo, Bosnia & Herzegovina 


Aida Kulo 

Institute of Pharmacology, Clinical Pharmacology 

and Toxicology, 

Faculty of Medicine, University of 

Sarajevo, 

Čekaluša 90, 71000 Sarajevo, Bosnia and 

Herzegovina 

Phone: +387 61 56 61 69; 

fax: +387 33 441 813; 

E-mail: aidakulo@yahoo.com 

Original submission: 

11 January 2010; 

Revised submission: 

14 March 2010; 

Accepted: 

13 April 2010. 

ABSTRACT 


Aim To evaluate differences in the treatment quality between often 

used oral anticoagulants, warfarin and acenocoumarol in patients 

with nonvalvular atrial fibrillation (NVAF). 

Methods This was an observational, comparative, one-year clinical 

study, conducted in the Blood Transfusion Institute of Sarajevo, 

Bosnia & Herzegovina. All patients who were using warfarin/ 

acenocoumarol and monitored were eligible. Patients who met 

inclusion criteria (the age of 40-80, diagnosed NVAF, CHADS 2 

index score ≥2, the planned long-term treatment) were included 

in two parallel groups of 60 patients, composed according to the 

warfarin/acenocoumarol treatment as well as the gender and age. 

Routinely measured International normalised ratio (INR) values 

were the basic parameter for individual quality and stability assessment. 

Results All average, monthly INR values were in therapeutic range 

(2.0-3.0) in both therapeutic groups. There were no significant 

differences either in the number of therapeutic INR values per patient 

(50.53±23.72% vs. 51.74±26.68%, P = 0.795) or in individual 

quality of treatment: >50% therapeutic INR values (60.0% 

vs. 64.9%, P = 0.721) and >75% therapeutic INR values (18.3% 

vs. 22.8%, P = 0.714) in the warfarin and acenocoumarol group, 

respectively. Significantly better stability was determined for acenocoumarol 

as compared with warfarin treatment in terms of a 

longer period of the total observed time during which therapeutic 

INR values were stable (37.6% vs. 35.7%, P = 0.0002). 

Conclusion Both drugs have shown similar quality of individual 

anticoagulation control, but acenocoumarol have shown significantly 

better anticoagulation stability with therapeutic INR values 

covering significantly longer time of treatment. 

Key words: nonvalvular atrial fibrillation, warfarin, acenocoumarol, 

INR, stability 

9

10 


INTRODUCTION 

Despite the continuously growing number of atrial 

fibrillation patients (1) and increased possibility 

of development of new ones, more specific and 

potentially more stable anticoagulants, the usage 

of old oral anticoagulants, warfarin and acenocoumarol 

is still increasing (2,3). These drugs are 

in the top 100 of the most prescribed drugs in the 

world (4). The increased usage of these drugs was 

noted from the early nineties until today, especially 

in the North America and Western Europe 

(5). Comparative studies of vitamin K antagonists, 

warfarin and acenocoumarol are rare, probably 

due to their geographical distribution according 

to which certain regions use only warfarin 

while other regions use only acenocoumarol (6). 

Warfarin is the first line drug for North America, 

Scandinavia and Great Britain. Although unavailable 

in the United States of America, most European 

countries, including Germany, have acenocoumarol 

as the first line drug (7,8). In other parts 

of Europe and elsewhere, acenocoumarol is often 

used as an alternative drug to warfarin. 

Due to its longer elimination half-life (30-80 hours, 

mostly around 37 hours), most of the authors 

(9-13) consider warfarin treatment to be better 

and more stable as compared to acenocoumarol. 

Yet, there are studies that deny this hypothesis 

(14-17). The narrow therapeutic index of vitamin 

K antagonists, next to multifactorial variations, 

creates difficulties in maintaining optimal coagulation 

status which prevents thrombose and 

avoids bleeding (18-22). Maintenance of referent 

International normalised ratio (INR) values (2.0- 

3.0) is basic criteria of safety and effectiveness of 

oral anticoagulant treatment (3,23,24). 

The aim of this study was to compare the individual 

quality and stability of long-acting warfarin 

and short-acting acenocoumarol in patients with 

nonvalvular atrial fibrillation (NVAF). After an 

analysis of the relevant databases we found this 

study to be the first comparative study of the oral 

anticoagulant treatment in Bosnia and Herzegovina, 

where patients have both drugs available 

PATIENTS AND METHODS 

Study design 

This was an observational, comparative, bidirectional 

(prospective/retrospective) one-year 

clinical study, conducted in the Blood Transfusion 

Institute Sarajevo, Bosnia & Herzegovina. 

The study was conducted according to the GCP 

(Good Clinical Practice), GLP (Good Laboratory 

Practice) and local ethical principles. 

Patients 

All patients with atrial fibrillation (AF) who 

were using warfarin/acenocoumarol and were 

previously monitored by the Blood Transfusion 

Institute were eligible. In the three-month period, 

213 patients were screened and 137 patients 

met inclusion criteria (age of 40-80, diagnosed 

NVAF, CHADS 2 index score ≥2 (25), the planned 

long-term treatment with warfarin/acenocoumarol 

which started at least 2 months prior to the 

observational period). The diagnosis of NVAF, 

indication for oral anticoagulant treatment and 

target INR range were determined in competent 

specialised institutions and adequately documented 

by medical records. The patients were divided 

into two parallel groups of 60 patients. In 

order to avoid a bias, the groups were composed 

according to the anticoagulant therapy (warfarin/ 

acenocoumarol) as well as their gender and age. 

The duration of the observational period was 

12 months. The first six months from the day of 

screening were observed retrospectively, and the 

following six months prospectively. The control 

visits and blood sampling for INR control were 

performed on a monthly basis, or more frequently 

if necessary. 

Data collection 

Anamnestic data collected during the screening 

visit (age, gender, weight, height, CHADS 2 index, 

comorbidities, concomitant drugs, smoking 

and alcohol use) were collected in a study sheet. 

Retrospective data (dates of INR measurements, 

INR values and the daily warfarin/acenocoumarol 

doses) for six months period prior to enrolment 

were collected via retrospective chart 

review of monthly patient organisers and gathered 

in a specially designed Patient’s Diary. For 

the following six months, the same data, as well 

as additional data (adherence to the treatment, 

temporary or permanent interruptions of the treatment 

and adherence to the low vitamin K intake 

diet) were assessed and collected at each visit 

prospectively.

Figure 1. Monthly average INR values on warfarin and acenocoumarol 

treatment in one-year observational period 

INR measurement 

INR values were measured routinely in the same 

laboratory. The blood samples were taken by the 

venipuncture of the cubital vein into a 3.2 sodium 

citrate test-tube (Vacutainer, Becton Dickinson, 

Great Britain). Fresh plasma obtained from blood 

samples was centrifuged at room temperature 

at 3000 g for 15 minutes (Laboratory centrifuge 

CENTRIC 322A, Slovenia). The measurement of 

INR values was done according to the following 

formula: INR = (PT ratio) ISI (International Sensitivity 

Index) (26). The automatic hemostasis 

scanner was used (CD-X, Switzerland), with 

liquid calcium rabbit thromboplastin (DiaPlastin-E, 

ISI=1.1). 

Statistical analysis 

A statistical analysis was performed by MedCalc 

for Windows program, Version 10.1.2.0 (Med- 

Calc Software, Mariakerke, Belgium). The comparison 

of the overall quality of treatment expressed 

by the percentage of therapeutic INR values, 

was performed by applying the chi-square test. 

The comparison of the individual quality of treatment, 

expressed by percentage of therapeutic 

INR values per patient in group, was performed 

by using t the test. Percentages of patients with 

more than 50% and with more than 75% of therapeutic 

INR values were compared among groups 

using the chi-square test. Stability of treatment, 

expressed by percentages of time during which 

therapeutic INR values were stable, was compared 

among groups using the chi-square test. An 

INR is defined as stable if it remains in the referent 

range within 2 consecutive measurements 

having at least 3 weeks period between them 

(27). A P value of less than 0.05 was considered 

statistically significant. 

Kulo et al Oral anticoagulant treatment stability 

Figure 2. Distribution of subtherapeutic, therapeutic and 

supratherapeutic INR values on warfarin and acenocoumarol 

treatment in one-year observational period 

RESULTS 

A total of 60 patients in the warfarin and 57 patients 

in the acenocoumarol group were observed. 

Since they have changed their anticoagulant 

therapy, three patients from the acenocoumarol 

group were excluded from the study. Homogenisation 

of the therapeutic groups according to demographical 

characteristics is presented in Table 

1. The groups were also composed by comorbidities, 

concomitant drugs, smoking and alcohol 

use, the adherence to the treatment, temporary or 

permanent interruptions of the treatment and the 

adherence to the low vitamin K intake diet. 

A total of 649 and 608 INR measurements were 

performed in one year period, with average time 

intervals between 33.35 ± 8.72 and 27.17 ± 11.37 

days in the warfarin and acenocoumarol group, 

respectively. All monthly average INR values 

were in the therapeutic range (2.0-3.0) in both 

therapeutic groups so differences between groups 

were clinically irrelevant (Mann-Whitney test, P 

= 0.0011) (Figure 1). 

There were no significant differences in the overall 

quality of the treatment expressed by percentage 

of therapeutic INR values (51.77% vs. 

53.62%, P = 0.548) in warfarin and acenocoumarol 

group, respectively. The distribution of therapeutic, 

subtherapeutic and supratherapeutic INR 

values has shown that the largest amount of INR 

measurements had the value of 1.5-2.5, bearing 

in mind that the therapeutic INR values of 2.0-2.5 

covered approximately 36% of all measurements 

in both therapeutic groups (Figure 2). 

In respect to individual quality of treatment, there 

were no significant difference in the mean percentage 

of therapeutic INR values per patient 

(50.53 ± 23.72% vs. 51.74 ± 26.68%, P = 0.795) 

in the warfarin and acenocoumarol group, respec- 

11

12 


TABLE 1. The comparison of demographic characteristics and 

other factors influencing the oral anticoagulant treatment in 

warfarin and acenocoumarol group 

Variable* 

tively. By applying the chi-square test, no significant 

differences were found in the percentage 

of patients with more than 25% therapeutic INR 

values (13.3% vs. 19.3%, P = 0.538), the percentage 

of patients with more than 50% therapeutic 

INR values (60.0% vs. 64.9%, P = 0.720) and 

the percentage of patients with more than 75% 

therapeutic INR values (18.3% vs. 22.8%, P = 

0.714), in the warfarin and acenocoumarol group, 

respectively (Table 2). The last represent high quality 

control of anticoagulant treatment. 

The stability of treatment, defined as the percentage 

of time during which therapeutic INR values 

were stable, was significantly better in acenocoumarol 

as compared with warfarin group (35.73% 

vs. 37.62%, χ2 = 14.012, P = 0.0002, Table 3) 

according to the chi-square test. 

DISCUSSION 

Warfarin 

X±SD 

Acenocoumarol 

X±SD 

t-test 

P † 

Age 66.17 ± 8.37 68.05 ± 8.59 > 0.05 

Gender M 55% / F 45% M 56% / F 44% > 0.05 

Employment 

status 

E 5 / R 55 E 2 / R 55 > 0.05 

Height (cm) 172.40 ± 7.81 172.00 ± 8.47 > 0.05 

Weight (kg) 81.67 ± 14.09 78.95 ± 12.49 > 0.05 

BMI§ (kg/m2 ) 27.47 ± 4.51 26.65 ± 3.53 > 0.05 

BSA (m2 ) 1.97 ± 0.20 1.94 ± 0.18 > 0.05 

No of comedications 

4.62 ± 1.47 5.04 ± 1.64 > 0.05 

CHADS score 2 2.58 ± 0.83 2.42 ± 0.63 > 0.05 

*Employment status: E, employed; R, retired; BMI, Body Mass Index; 

BSA, Body Surface Area; CHADS, congestive heart disease, hypertension, 

age, diabetes mellitus, stroke; .†significant, P < 0.05, t-test 

The reliability of our results was strengthened by 

the homogenisation of therapeutic groups based 

on the number of patients, gender and age, as 

well as homogenisation according to other significantly 

important factors that are known to have 

TABLE 2. Comparison of individual quality of treatment in 

warfarin and acenocoumarol group 

% of 

therapeutic 

INR* 

values 

No (%) of patients χ 2 test 

Warfarin Acenocoumarol P † 

< 25% 8 (13.3) 11 (19.3) 0.5328 

25-50% 16 (26.7) 9 (15.8) 0.6624 

> 50% 36 (60.0) 37 (64.9) 0.7208 

> 75% 11 (18.3) 13 (22.8) 0.7144 

*INR, International normalised ratio; *significant, P < 0.05, chisquare 

test 

an impact on anticoagulant treatment. The results 

of our study have shown that all monthly average 

INR values within one year were in therapeutic 

range (2.0-3.0) in both therapeutic groups 

with clinically irrelevant differences between the 

groups; the average INR values in comparisson 

with warfarin treatment in acenocoumarol group 

were 91.6% of all measurements closer to the target 

INR value of 2.5. An average time interval 

between the two consecutive measurements registered 

in both therapeutic groups in our study 

was much higher than around 19 days (28), and 

lower than around 32 days (29). Patients using 

acenocoumarol required in average less frequent 

INR measurements in our study, while the results 

of other studies have shown that a lower number 

of measurements per patients were registered on 

warfarin treatment (9, 10). 

There was no significant difference in the quality 

of treatment for warfarin and acenocoumarol 

(51.77% and 53.62%, respectively) which is similar 

to findings of other studies (17) (65.5%, 63.4%, 

respecively, P > 0.05), and the acenocoumarol can 

be applied with the similar treatment quality (14). 

According to one retrospective case control research, 

the overall quality of treatment was significantly 

better in the warfarin group (72% vs. 67%, 

P < 0.001), which is opposite to our results (9). 

Warfarin is also suggested as the first line drug for 

NVAF according to results of a multicentric retrospective 

study (29). On the other hand, other studies 

have shown that the overall quality of the acenocoumarol 

treatment was significantly better in comparison 

with warfarin (56 ± 26.8% vs. 49 ± 22.6%, P 

< 0.05) (16). There are some results showing better 

(59%) (29) and some showing lower (36.5%, 38%) 

(6, 30) overall quality of acenocoumarol treatment 

in comparision with our study. 

Upon the comparison of individual quality of treatment, 

the results of our study have not shown 

significant differences either in the percentage of 

patients with more than 50% therapeutic INR values 

or in the higher quality with more than 75% 

therapeutic INR values for warfarin and acenocoumarol. 

The results related to the percentage of pati- 

TABLE 3. Comparison of warfarin and acenocoumarol treatment 

stability 

Treatment No (%) of observed treatment days χ 2 

test 

Warfarin Acenocoumarol P* 

stable 

treatment 

6670 (35.73) 6768 (37.62) 0.0002 

*significant, P < 0.05, chi-square test

ents having more than 50% therapeutic INR values 

were superior as compared to the results of most 

other studies with significance in favour of warfarin 

(6,9,17). On the other hand, considerably better 

individual quality of treatment in acenocoumarol 

group in comparison with our results have been 

registered (29). A significantly higher percentage 

of patients with more than 75% therapeutic INR 

values on warfarin in comparison with acenocoumarol 

treatment have also been shown (9). 

Our results have pointed out the important advantage 

of acenocoumarol regarding the better 

stability of oral anticoagulant treatment. Namely, 

comparing the total number of treatment days of 

all patients in the group, acenocoumarol in the 

comparison with warfarin ensured significantly 

longer period of stability maintenance of therapeutic 

INR values. It has been confirmed by other 

authors (11). On the other hand, similar stability 

of warfarin and acenocoumarol treatment 

was also registered (15), while other studies have 

registered the expected advantage of warfarin 

(16). Based on research of risk instability factors 

of oral anticoagulant treatment, it has been concluded 

that the OR of instability were higher for 

the working people when compared with retired 

ones, higher for acenocoumarol in comparison 

with warfarin and phenprocoumon and for noneducated 

in comparison with patients that are 

familiar with the reasons and clinical importance 

of oral anticoagulant therapy (12,15). Still, in 

comparison with other studies, maintenance of 

stable therapeutic INR values was shorter in our 

study. Time in which INR values remain stable 

in randomised clinical studies was between 44 

and 83% (31). The stable INR values were maintained 

in acenocoumarol treatment in 41.6% (6), 

43% (30) and 64.9% of time (29). 

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Elastic characteristics of aorta in patients with epilepsy 

Namık Ozmen 1 , Mehmet Senol Guney 2 , Erdem Togrul 2 , Omer Yiginer 1 , Mustafa Aparci 1 , Ejder Kardesoglu 

1 , Yılmaz Cingozbay 1 , Mehmet Saracoglu 1 , Bekir Sitki Cebeci 1 

1 Department of Cardiology, 2 Department of Neurology; GMMA Haydarpasa Trainning Hospital, Istanbul, Turkey 

Correpanding author: 

Namık Ozmen 

GATA Haydarpaşa Eğitim Hastanesi 

Kardiyoloji Servisi 

34668 Kadıkoy Istanbul, Turkey 

Phone: + 216 542 20 20/ 3485; 

fax: + 216 3454881 

E-mail: drnamikozmen@yahoo.com 


04 January 2010; 


14 March 2010; 

Accepted: 

08 April 2010. 

ABSTRACT 


Aim To investigate elastic characteristics of the aorta in patients 

with epilepsy. 

Methods Seventy five patients with a diagnosis of epilepsy through 

clinical and EEG findings and age and sex matched, 50 healthy 

controls were included. Systolic and diastolic blood pressures plus 

systolic and diastolic diameter of the aortic root was measured. 

Aortic strain (AS) and aortic distensibility (AD) and aortic distensibility 

index (BSI) were calculated. 

Results The average age of the epilepsy group was 23.8.8 ± 8.2 

years, and of the control group it was 24.1± 6.2 years (p>0.05). AS 

and AD were lower in the epileptic group while the aortic stiffness 

index was higher (10.4± 4.2 vs 16.9±0.2, p: 0.001, for AS; 8.7± 

4.0 vs 17.2±0.1, p: 0.001, for AD and 20.1±0.1 vs 3.5±1.2, p: 

0.001 for BSI). 

Conclusion Elastic characteristics of the aorta change in epileptic 

patients, with a decrease of the distensibility of the aorta and an increase 

of the stiffness. After this preliminary study, new controlled 

studies are needed. 

Key words: Epilepsy, aortic stifness, atherosclerosis 

15

16 


INTRODUCTION 

Epilepsy is a clinical condition characterized by 

disturbances of consciousness and convulsive seizures 

and is associated with a disturbance of electrical 

activity of the brain. Its clinical spectrum is 

between simple or focal seizures to life-threatening 

serious status epilepticus (1). Although the 

lesion causing the seizures cannot be shown in 

every case, the main pathology is in the brain (2). 

Actually epilepsy is not a single disease. A better 

definition would be a group of different syndromes 

and diseases with a multitude of different 

manifestations and causes. Some of these causes 

are the main factor initiating the seizures, while 

others may be contributing factors (1,2). 

In some cardiac pathologies (e.g. syncope, AV 

blocks long QT syndrome) the cerebral perfusion 

may be affected and this may contribute to or 

provoke the development of some seizures (3,4). 

Some patients with cardiovascular diseases may 

present with epilepsy-like clinical pictures. Cases 

who have been followed up and had received treatment 

for epilepsy whose cardiac pathology has 

been missed have also been reported (5). In some 

of these patients, after treating the cardiac pathology, 

antiepileptic therapy could be stopped (3,5). 

Arterial stiffness is the result of elastic tissue loss 

in the vessel wall (6). This suppresses some of 

the pulsations from the heart and the non-stop 

blood flow in the artery (6). Due to the structural 

changes caused by cardiovascular risk factors, 

the vessels become stiffer and the compliance 

decreases. It has been shown that with the 

increase of arterial stiffness in large vessels, 

the cardiovascular mortality increases (7,8). Increased 

aortic stiffness is an independent risk factor 

for prognosis in individuals with chronic artery 

disease (9). Arterial stiffness develops from 

changes in dynamic and complex interactions of 

the cellular and structural elements in vessel wall 

(6,7). Glucose regulation, salt, and sympathetic 

activation can cause these vascular changes (10). 

It is known that changes in sympathetic activation 

occur before and after epileptic seizures, and 

thus changes in blood pressure, heart rate and 

cardiac output occur (10,11). Increased sympathetic 

activation before or just after a seizure may 

lead to vascular injury in long term. There is no 

data on the elasticity of the aorta in epileptic patients. 

We hypothesized that aortic stifness could 

be increased in patients with epilepsy and in this 

study we tried to address this issue. 

METHODS 

Seventy five subjects with a diagnosis of epilepsy 

through clinical and EEG findings were included 

in the study. As a control group, age and sex matched, 

50 healthy individuals were included. All 

individuals in the study were informed about the 

study and their written consent was taken. The 

local ethical committee approved the study. 

The EEG and echocardiographic evaluation was 

made by blinded neurologist and cardiologist. All 

epileptic individuals were referred to the neurology 

and cardiology policlinics. All these individuals 

had been diagnosed with epilepsy and had 

been followed up for at least two years prior to 

the study, all were literate and all were above 18 

years of age. None had a cardiac problem at the 

time of the study. The sociodemographic features, 

including how long they had the disease, the 

medications used and the presence of individuals 

with cardiac diseases in their relations were noted. 

Electrocardiogram (ECG) and echocardiograms 

were obtained and cardiac examination was done. 

The echocardiagraphic evaluation was made with 

VingMed System FiVe device (GE Medical System, 

Hortan Norway) with 2.5 MHz probe while the patient 

was lying on his/her left side, breathing, and 

using standard methods. Throughout the evaluation 

the echocardiographic follow-up was done using 

the ECG channels. In the initial echocardiographic 

evaluation, the presence of pathology was ruled out 

using two dimensions, colored Doppler, pulsed and 

continuous wave Doppler in the parasternal long 

axis, short axis, apical 4 and 2 chamber images and 

standard methods. Afterwards, M mode echocardiagraphic 

records were obtained from the parasternal 

long axis. The measurements were made and 

specifically the left ventricule ejection fraction (EF) 

was recorded. The same image was modified and 

ascending aorta was visualized. M mode echocardiographic 

records of the aorta 3 cm above the aortic 

valve were obtained. Systolic (AoS) and diastolic 

(AoD) diameter of the aorta was measured from the 

recordings. AoS was measured at the end of systole 

when the aortic valve was fully open and AoD was 

measured at the time of QRS peak at the ECG and 

these measurements were recorded (Figure 1). At 

least three measurements were made from each in-

dividual and an average of these measurements was 

used in the formulae. 

Aortic Strain (AS), aortic distensibility (AD) and 

beta stiffness index, which show the elasticity of 

the aorta, were calculated using the following 

formula (12). 

AS (%)=100 x (AoS-AoD)/AoD 

AD (cm-2 dyn-1 10-6 )=2 x AS x (SBP-DBP) 

BSİ= (SBP/DBP)/ (SBP-DBP) / AoD 

None of the patients with any known systemic, 

cardiac or neurologic disease other than epilepsy 

had been included in the study. 

The statistical analysis was performed using a paired 

Student’s t test for continuous variables with 

the SSPS Statistical Package, Windows version 

(SSPS 13, Chicago, USA). In this study, p values 

lower than 0.05 were considered statistically significant. 

All data were expressed as mean ± SD 

(Standard deviation). 

RESULTS 

The average age of the patients in epilepsy group 

was 23.8.8 ± 8.2 years, and of the patients in control 

group was 24.1± 6.2 years (p>0.05). In both groups 

8 % of patients were female (10 patients and 4 

controls). Thus the average age and sex was similar 

in both groups. The systolic and diastolic blood pressures 

were similar between the groups. The systolic 

and diastolic diameters in the aortic root were higher 

in the epilepsy group. Similarly the aortic strain and 

distensibility was lower in the epileptic group while 

the beta stiffness index was higher (Table 1). 

DISCUSSION 

We detected that in epileptic patient systolic 

and diastolic diameters of aortic root were lar- 

Figure 1. Measurement of systolic and diastolic diameter 

of aortic root (S, Systolic diamter of aortic root; D, Diastolic 

diameter of aortic root) (N. Ozmen, 2009) 

Ozmen et al Aortic distensibility in epilepsy 

ger than in the control group. Similarly, both the 

aortic strain and distensibility were lower in the 

epileptic group while the beta stiffness index was 

higher than in the control group. 

Aortic stifness is a result of aging and known 

atheroslerotic risk factors like smoking, hypercholesterolemia, 

diabetes mellitus, hypertension 

(13-15). Increased aortic stifness is accepted as 

predictor of atherosclerotic changes in vascular 

system (16,17). 

Epilepsy is a common paroxysmal disease, with 

an incidence of 0.4-0.7 % in developed and 1-1.9 

% in developing countries (5). As discussed above, 

etiology can be detected in one-to-third or 

one-to-quarter patients. The other patients are named 

as idiopathic or cryptogenic (18). 

Aortic stiffness is related to reduction in elastine 

component of aortic root because of a different 

disease. The store volume (the blood remaining 

after the diastole) is dependent on arterial compliance. 

A great volume can be stored in the aorta 

and great arteries during systole. Thus, the greatest 

share on the arterial circulation compliance is 

in the proximal aorta and its branches (19). With 

an increase of the aortic stiffness, there is an increase 

of atherosclerosis, cardiovascular morbidity 

and mortality (13,16). 

Various studies have shown that aortic stiffness 

increases in the stroke population (20). Also the 

carotid artery distensibility is reported to increase 

in patients with ischemic stroke (21). Also again, 

Nemes et al (2008) have reported an increase of 

aortic stiffness in patients with hereditary optic 

neuropathy (22). Our study is different with respect 

to inclusion of patients with epilepsy. 

The factors contributing to arterial compliance 

include the structure, size and function of the 

artery, blood volume, pressure in the artery and 

Table 1. The data of epilectic patients and control group* 

Parameter 

Epilepsy 

(n=75) 

Control 

(n=50) 

P value 

Age (years) 23.8 ± 8.2 24.1 ± 6.2 > 0.05 

SBP (mmHg) 119.1 ± 6.5 117.7 ± 4.2 0.07 

DBP (mmHg) 76.5 ± 4.7 77.2 ± 1.3 0.5 

AoS (mm) 31.1 ± 2.8 28.3 ± 2.6 0.001 

AoD (mm) 28.1 ± 2.5 25.2 ± 0.2 0.001 

AS (%) 10.4 ± 4.2 16.9 ± 0.2 0.001 

AD (cm-2 dyn-1 10-6 ) 8.7± 4.0 17.2±0.1 0.001 

BSI 20.1±0.1 3.5±1.2 0.001 

*SBP, systolic blood pressure; DBP, diastolic blood pressure; AOS, 

aorta root systolic diameter; AOD, aorta root diastolic diameter; AS, 

aortic strain; AD, aortic distensibility; BSI, beta stifness index 

17

18 


autoregulatory mechanisms (7,8). It has been 

reported that epileptic seizures decrease in individuals 

practicing yoga (23). This suggests the 

contribution of autonomic nervous system. It is 

known that the changes in sympathetic activation 

occur before and after epileptic seizures, and thus 

changes in blood pressure, heart rate and cardiac 

output occur. Also, this situation might be in relation 

to mortality in epileptic patients and rats 

(23,24). Therefore, it is theoretically possible that 

changes in the elastic properties of the aorta may 

occur from haemodinamyc changes during or 

after seizures. Arteryal stiffness has significant 

prediction in patients with heart failure, stoke, 

REFERENCES 

1. Rocamora R, Kurthen M, Lickfett L, Von Oertzen J, Elger 

CE. Cardiac asystole in epilepsy: clinical and neurophysiologic 

features. Epilepsia 2003; 44:179–85. 

2. Devinsky O, Pacia S, Tatambhotla G. Bradycardia and 

asystole induced by partial seizures: a case report and 

literature review. Neurology 1997; 48:1712–4. 

3. Jallon P. Arrhythmogenic seizures. Epilepsia 1997; 38 

(suppl 11):S43–7. 

4.Lim E CH, Lim S-H, Wilder-Smith E. Brain seizes, heart 

ceases: a case of ictal asystole. J Neurol Neurosurg 

Psychiatry 2000; 69:557–9. 

5. Hunt DP, Tang K. Long QT syndrome presenting as 

epileptic seizures in an adult. Emerg Med J 2005; 

22:600-1. 

6. Demiralp E, Kardesoglu E, Kiralp MZ, Cebeci BS, Keskin 

I, Ozmen N, Dursun H. Aortic elasticity in patients 

with ankylosing spondylitis. Acta Cardiol 2004; 

59:630-4. 

7. Laurent S, Boutouyrie P, Asmar R, Gautier I, Laloux B, 

Guize L, Ducimetiere P, Benetos A. Aortic stiffness is 

an independent predictor of all-cause and cardiovascular 

mortality in hypertensive patients. Hypertension 

2001; 37:1236-41. 

8. Stefanadis C, Dernellis J, Tsiamis E, Stratos C, Diamantopoulos 

L, Michaelides A, Toutouzas P. Aortic 

stiffness as a risk factor for recurrent acute coronary 

events in patients with ischemic heart disease. Eur 

Heart J 2000; 21:390-6. 

9. Madhavan S, Ooi WL, Cohen H, Alderman MH. Relation 

of pulse pressure and blood pressure reduction 

to the incidence of myocardial infarction. Hypertension 

1994; 3:395-401. 

10. Xu C, Zarins CK, Pannaraj PS, Bassiouny HS, Glagov 

S. Hypercholesterolemia superimposed by experimental 

hypertension induces differential distribution 

of collagen and elastin. Arterioscler Thromb Vasc 

Biol 2000; 20:2566-72. 

11. Metcalf CS, Radvanski PB, Bealer SL. Status epilepticus 

produces chronic alterations in cardiac sympathovagal 

balance. Epilespsia 2009; 50:747-54. 

12. Stefanidis C, Wooley CF, Bush CA, Kolibash AJ, Boudoulas 

H. Aortic distensibility abnormalities in coronary 

artery disease. Am J Cardiol 1987; 59:1300-4. 

renal failure and previous myocardail infarction 

too (23,24). We could not find any other study 

investigating the elastic properties of the aorta in 

epilepsy patients and our study is the first one on 

this issue in the literature . 

In conclusion, it appears that the elastic characteristics 

of the aorta may change in epileptic 

patients, with a decrease of the distensibility of 

the aorta and an increase of the stiffness. But we 

think that more studies are needed in relation to 

this subject. 



13. Metcalf CS, Poelzing S, Little JG, Bealer SL. Status 

epilepticus induces cardiac myofilament damage and 

increased susceptibility to arrhythmias in rats. Am J 

Physiol Heart Circ Physiol 2009; 297:H2120-7. 

14. Franklin SS, Larson MG, Khan SA, Wong ND, Kannel 

WB, Levy D. Does the relation of blood pressure 

to coronary heart disease risk change with aging? 

The Framingham Heart Study. Circulation 2001; 

103:1245–9. 

15. Vaccarino V, Berger A, Abramson J, Black H, Setaro J, 

Davey J, Krumholz H.Pulse pressure and risk of cardiovascular 

events in the systolic hypertension in the 

elderly program. Am J Cardiol 2001; 88:980–6. 

16. Benetos A, Safar M, Rudnichi A, Smulyan H, Richard, 

J-L, Ducimetie`re P,Guize L. Pulse pressure: a predictor 

of long-term cardiovascular mortality in a french 

male population. Hypertension 1997; 30:1410 –5. 

17. Galis ZS, Khatri JJ. Matrix metalloproteinases in vascular 

remodeling and atherogenesis. Circ Res 2002; 

90:251–62. 

18. Akalın F, Tırtır A, Yılmaz Y. Increased QT dispersion 

in epileptic children Acta Pediatr 2003; 92:916-20. 

19. Lee RT, Kamm RD.Vascular mechanisms for the cardiologists. 

J Am Coll Cardiol 1994; 23:1289-95. 

20. Lehmann ED, Hopkıns KD, Jones RL, Rudd AG, 

Goslıng RG. Aortic distensibility in patients with cerebrovascular 

disease. Clin Sci 1995; 89:247-53. 

21. Harloff A, Strecker C, Reinhard M, Kollum M, Handke 

M, Olschewski M, Weiller C, Hetzel A. Combined 

measurement of carotid stiffness and intima-media 

thickness improves prediction of complex aortic 

plaques in patients with ischemic stroke. Stroke 2006; 

37:2708-12 

22. Nemes A, De Coo IF, Spruijt L, Smeets HJ, Chinnery 

PF, Soliman OI, Geleijnse ML, Ten Cate SJ. Is there 

alteration in aortic stiffness in Leber hereditary optic 

neuropathy? Eur J Ophthalmol 2008; 18:309-12. 

23. Sathyaprabha TN, Satishchandra P, Pradhan C, Sinha 

S, Kaveri B, Thennarasu K, Murthy BT, Raju TR. 

Modulation of cardiac autonomic balance with adjuvant 

yoga therapy in patients with refractory epilepsy. 

Epilepsy Behav 2008; 12:245-52. 

24. Benetos A, Laurent S, Hoeks AP, Boutouyrie PH, Safar 

ME. Arterial alterations with aging and high blood 

pressure. A noninvasive study of carotid and femoral 

arteries. Arterioscler Thromb 1993; 13:90 –7.


Predictive value of fetal nuchal translucency 


Department of Gynecology and Obstetrics, Kragujevac Clinical Center, Kragujevac, Serbia 



Department of Gynecology and 

Obstetrics, Kragujevac Clinical Center 

Vojislava Kalanovića 1A /3, 

34000 Kragujevac, Serbia 

Phone: +381 64 616 8999; 

fax: + 381 34 370 151 

Email: drloncar@sezampro.rs 


20 March 2010; 


23 April 2010; 

Accepted: 

24 August 2010. 

ABSTRACT 


Aim To determine the predictive value of fetal nuchal translucency 

(NT) measurements in the detection of chromosomopathy. 

Methods The investigation of NT included 317 pregnant women 

with singleton pregnancies of gestational age of 11 to 14 weeks 

with pathological karyotype. The control group consisted of pregnant 

women whose amniocentesis findings indicated a normal 

fetal karyotype. 

Results The median value of NT in the control group was 1.92 

± 0.39 mm, and in the group with pathological fetus karyotype 

findings it was 2.49 ± 0.37 mm (p 0.05). The probability for a patient with negative NT findings 

to be healthy was 1.0, NT sensitivity as a marker for chromosomopathy 

was 1.0. The rate of false positive findings was 0.026. The 

specificity of NT as a marker for chromosomopathy was 0.97. 

Conclusion Normal NT findings could be considered reliable ultrasonographic 

markers in the assessment of the absence of chromosomopathy. 

Pathological findings, given the low positive predictive 

value of NT must be interpreted in the context of other 

prenatal tests before the pregnant woman is advised to undergo 

invasive prenatal diagnosis. 

Keywords: nuchal translucency, ultrasonography, chromosomopathy, 

predictive statistics 

19

20 


INTRODUCTION 

In antenatal protection - monitoring growth and 

development of an unborn child, in most European 

countries it is recommended to perform three ultrasound 

scans: at 9 - 12, 19 - 22 and 29 - 32 weeks 

(1). In case of any abnormalities or complications 

of pregnancy, additional ultrasound scans could 

provide additional safety to pregnant women and 

gynecologists monitoring the pregnancy. In search 

for specific early ultrasound signs - markers that 

could indicate an increased risk of hereditary or 

acquired disorders (fetal chromosomopathies), scientific 

studies have confirmed an exceptional value 

of nuchal fold ultrasound findings (nuchal translucency, 

NT) (2). Nuchal fold scan measures fluid 

accumulation between the skin and subcutaneous 

fascia in the neck of an embryo. It is performed 

between 11 and 14 weeks of pregnancy, or when 

the crown- rump length (CRL) is 45 to 84 mm (3). 

Fold thickness of less than 99 percentile for CRL 

is usually tolerated (4). Numerous studies have 

shown association between ultrasound findings 

of nuchal fold > 3 mm and specific chromosomal 

aberrations, especially with aneuploidy and Down 

syndrome (5). The correlation of NT scan findings 

with Down syndrome is of such importance that 

most authors consider nuchal fold scan a screening 

procedure for Down syndrome (6). In the study of 

King’s group which was done at over 96,000 pregnancies 

(22 perinatal centers, 306 gynecologists) 

NT scan has indicated Down syndrome in 82% 

of fetuses (frequency of false positives: 8.3%) 

(7). In addition to association with chromosomal 

aberrations, nuchal fold thickness is also used as 

a marker for other genetic syndromes, usually heart 

anomalies (8). Fetal NT increases with CRL, 

therefore, it is very important to take into account 

gestation period when determining whether the 

measured NT was increased or not (9). In a study 

that involved 96,127 pregnancies, the NT median 

value and 95-percentile at a crown-rump length of 

45 mm were 1.2 and 2.1 mm; the respective values 

at a crown-rump length of 84 mm were 1.9 and 2.7 

mm (10). In pregnancies with fetal NT below the 

99th percentile (3.5 mm), parents’ decision regarding 

the determination of the fetal karyotype depended 

on the individual risk, accessed based on 

maternal age, ultrasound findings and free β-HCG 

and PAPP-A in maternal serum between 11-13 +6 

weeks (11). 

Several prospective interventional studies have 

examined implementation of NT screening into 

routine clinical practice (12). In our clinic in Kragujevac 

since 2008 we have reviewed nuchal fold 

thickness of fetuses in all pregnant women as an 

integral part of the review at the beginning of the 

second trimester of pregnancy. The resulting value 

was used to calculate the risk of using combined 

test. The aim of this study is to determine the 

predictive value of fetal NT measurements in the 

detection of chromosomopathy. 


The study was conducted at the Clinic for Gynecology 

and Obstetrics of the Clinical Center 

in Kragujevac (Serbia) on singleton intrauterine 

pregnancies in the first trimester of pregnancy 

in the period 2007-2009. A clinical experimental 

study model was used. All subjects read and 

signed informed consent for participation in this 

study. Ethical Committee of the Clinical Center 

Kragujevac approved the study. 

The investigation included 317 pregnant women 

with singleton pregnancies monitored by Genetic 

Counselling Service Committee of the Clinic 

of Gynaecology and Obstetrics of the Clinical 

Center Kragujevac. The study inclusion criteria 

involved the crown-rump length (CRL) between 

45 and 84 mm and the gestational age of 11-13+6 

weeks. For measurements of fetal nuchal translucency 

thickness (NT) a high-resolution ultrasound 

device Aloka Pro Sound 3500 with a cine-loop 

function was used, which allowed replay and 

a caliper providing measurements to 1 decimal 

point. Only the fetal head and the upper thorax 

were scanned. Since the magnification was maximal, 

each slight movement of the caliper produced 

only a 0.1 mm change. The nuchal translucency 

was measured with the fetus in a neutral 

position. The maximum thickness of the subcutaneous 

translucency between skin and the soft 

tissue overlying the cervical spine was measured. 

The calipers were placed on the lines defining 

the NT thickness so that they can hardly be seen 

on a white borderline behind the neck (Figure 

1). Several measurements during the scan were 

made, but only maximum measurement for the 

risk assessment was used. In cases where umbilical 

cord was around the fetal neck (in about 8% 

of the cases) the NT thickness above and below

the umbilical cord was measured and its average 

values were used. 

Having performed the early amniocentesis, the 

obtained karyotype results were divided into two 

groups as follows: pregnant women with numerical 

aberrations (SP) and those with structural disorders 

on the level of chromosomes (LP). Using 

contingency tables predictive value of the nuchal 

translucency (NT) was determined as a possible 

marker of invasive prenatal screening of pregnant 

women of gestational age 11 to 13 +6 Figure 1. Nuchal translucency measured with the fetus in 

neutral position (D. Lončar, 2010) 

weeks: positive 

predictive value (SP/SP+LP) (number of 

women with positive findings that have developed 

Table 1. Ultrasonography markers in the group of pregnant 

women with pathological karyotype results after early 

amniocentesis 

Protocol No 

Nuchal 

translucency 

(mm) 

(NT) 

Crown 

to rump 

length 

(CRL) 

(mm) 

Gestational 

age (GS) 

(days) 

Karyotype after 

early amniocentesis 

3-2007 2.2 60 86 46,xy/47xyy 

11-2007 3.0 62 88 

46,xx/46,xx; del 

7t(7;17) 

47-2007 2.5 65 88 47,xy +21 

151-2007 2.6 63 86 47xy+21 

74-2008 1.8 73 90 47, xy+18 

76-2008 2.4 72 89 

Robertson translocation 

45, xy,-14, 

-21 +t (14q;21q) 

158-2008 2.5 56 82 47, xx+21 

99-2008 2.6 65 87 

Robertson translocation 

45,xx,-14,- 

21+t (14q21q) 

161-2008 2.7 48 81 47, xx+21 

164-2008 2.0 50 81 

46,xy/46, y del(x) 

t(7;x)q35;q22) 

162-2008 1.9 48 78 46,xy/46,xy (-4q3) 

167-2008 3.1 48 80 47,xy+21 

231-2009 2.8 61 89 47,xx+21 

267-2009 2.6 71 91 47,xx+21 

237-2009 2.4 56 87 

46,xx/47,xx t (9;6) 

(q31;q14) 

271-2009 2.8 64 88 46, xy/47,xy+13 

Lončar Predictive value of fetal nuchal translucency 

Table 2. Median values and standard deviations of ultrasonographic 

parameters in the total sample 

Parameter 

Nuchal 

Translucency (mm) 

Crown to rump length 

fetus (mm) 

the disease) and negative predictive value (SN/ 

SN+LN) (the number of women with negative test 

findings that have not developed the disease). 

The probability for a patient with positive NT findings 

to have the disease or numeric aberrations 

was 0.5, the probability for a patient with negative 

NT findings to be healthy was 1.0. 

The sensitivity of NT measurement as a marker 

for chromosomopathy was determined according 

to the formula: SPP =SP/SP+LN= 1.0 

The false positive rate was determined according 

to the formula: SLP =LP/LP+SN= 0.026. 

The specificity of NT measurement as a marker 

for chromosomopathy was determined according 

to the formula: SSN =SN/SN+LP= 0.97. 

For statistical analysis parametric and non-parametric 

tests were used to determine the significance 

of statistical difference, t test, χ2 test, Fisher’s 

exact test and contingency tables for calculation 

of parameters of predictive statistics. 

RESULTS 

Pathological Control group 

karyotype (No =16) (No =311) 

SD 

2.49±0.37 

SD 

1.92±0.39 

Fetal chromosomal aberrations were found in 16 

pregnant women (Table 1). 

Median value of the CRL was 64.83±8.23 mm 

and 60.12±8.48 mm in the control group and 

in the group with pathological karyotype, respectively. 

Gestational age was 87.40±7.10 and 

85.69±3.98 days in the control and pathological 

group, respectively (p0.05 

Table 3. Numerical results of pregnant women - contingency 

table 

Test result Disease present Disease absent Total 

Positive 8 8 16 

Negative 0 301 301 

Total 8 309 317 

21

22 


Figure 2. Distribution of the nuchal translucency values (NT) in 

relation to the crown-rump length (CRL) in the total sample. 

Distribution of NT values in relation to the median 

in the control group (1.90 mm) was 135:301, 

or 55.15% above the median, or 44.85% below 

the median (Figure 2,3). 

The distribution of NT thickness values measured 

in relation to the median (2.55 mm) NT with 

pathological karyotype after invasive diagnostic 

was 11:5 or 68.75% above the median, or 31.25% 

below the median (Figures 4,5). 

The analysis of the total sample found 26 pregnant 

women with NT above the median of 2.55 

mm for the given CRL and 61.54% of fetal chromosomal 

aberrations were confirmed by invasive 

diagnostic methods (Figures 4, 5). 

DISCUSSION 

Fetal chromosomal aberrations were found in 16 

pregnant women, which could be explained by 

sample preselection (all pregnant women suspected 

of fetal annormalities). Fetal nuchal translucence 

(NT) increases with a rise in CRL, therefore 

it is very important to obtain correct CRL measurements 

(12). The analysis of the NT values distribution 

has shown that the values were distributed 

Figure 3. Distribution of the nuchal translucency values (NT) 

in relation to the gestational age (NG) in the total sample 

Figure 4. Distribution of nuchal translucency values with 

pathological karyotype (NT pat) as compared to the crownrump 

length (CRL pat) 

around the median (2.55 mm) with 44% NT values 

below and 56% above the median value, which 

is in agreement with quality control criteria established 

by the Fetal Medicine Foundation (FMF) 

(13). According to FMF criteria the gestational age 

should be between 11 and 13+6 weeks, and the 

CRL between 45 and 84 mm (14, 15). The distribution 

of fetal NT values for the given CRL in our 

study did not differ from the standard distribution 

described by the FMF. Therefore, our NT measurements 

were properly performed. Bаrclay measured 

NT in the sample of 11,281 pregnant women 

and found 118 inborn fetal anomalies with 

52 trisomy, 21, 71.2% of which had NT above 

the 95th percentile (3 mm) in the period between 

11 and 14 weeks gestation. In the same study, this 

number falls down to 56 % in the period between 

15 and 16 weeks gestation (16). Measuring NT in 

32,000 fetuses of gestational age between 11 and 

14 weeks Mаnni has found average age of the pregnant 

women was 32 years, in 5.1% fetuses NT 

was above the 95th percentile (3 mm) and 87.1% of 

which had a normal karyotype; out of 110 diagno- 

Figure 5. Distribution of nuchal translucency values with 

pathologic karyotype (NT pat) in relation to gestational age of 

the pregnancy (NG pat)

sed chromosomepathies 72 were trisomy 21 (17). 

These findings, regardless to the fact that there was 

no statistically significant difference in the values 

could have an effect on measurements of other ultrasonographic 

markers. Similar results could be 

found in the literature: in the study conducted in 

Great Britain (GB) the incidence of Down Syndrome 

was 2.1 ‰ per 1,000 live births, which is 

50% above of the national GB data (18). In our 

sample, in the group with pathological karyotypes 

68.75% of pregnant women had NT values above 

the median for the given CRL, which is in agreement 

with the results found in the literature (17,18). 

Analyzing the total sample, we found 26 pregnant 

women with NT above the median of 2.55 mm for 

the given CRL and 61.54% of fetal chromosomal 

aberrations were confirmed by invasive diagnostic 

methods. This means that our results comply with 

the standards given by the FMF (13). In our study, 

the NT sensitivity as a marker for chromosomopathy 

was 1.00, and the specificity was 0.97, which 

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A very high negative predictive value of 100% 

obtained in this study practically means that NT 

as a marker in prediction of chromosopathy could 

be considered as a “correctly negative” rather 

than a “falsely positive” parameter. Normal 

nuchal translucency finding could be considered 

as a reliable ultrasonographic marker in the assessment 

of the absence of chromosomopathy. Pathological 

findings should be interpreted in the 

context of other prenatal tests before the pregnant 

woman is advised to undergo invasive prenatal 

diagnosis. Patients should always be aware that 

measuring nuchal tarnslucency is a screening 

procedure rather than the establishment of a definite 

diagnosis. The diagnosis should be based 

on both an invasive intervention (gold standard) 

and fetal karyotype determination. 



11. Linskens IH, Spreeuwenberg MD, Blankenstein MA, 

van Vugt JM. Early first-trimester free beta-hCG and 

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KH. UK multicentre project on assessment of 

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15. Wald NJ, Rodeck C, Hackshaw AK, Walters J, Chitty 

L, Mackinson AM; SURUSS Research Group. 

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Uticaj biometeoroloških faza na incidencu suicida 

Vladimir Gajić¹, Dragan Milojević¹, Jasminka Smailagić², Nela \onović³, Suzana Matejić 4 , Sanja Gajić 5 

¹Zavod za hitnu medicinsku pomoć Kragujevac, ²Republički hidrometeorološki zavod Srbije, ³Institut za javno zdravlje u Kragujevcu, 

4 Medicinski fakultet Univerziteta u Kragujevcu, 5 Dom zdravlja Kragujevac; Kragujevac, Srbija 


Vladimir Gajić, 

Zavod za hitnu medicinsku pomoć 

Kragujevac, 

Lole Ribara 19, 34 000 Kragujevac, Srbija 

Phone: +381 34370090; 

fax: +381 34370276; 

E-mail: drgaja@sbb.rs 


11. novembar 2009.; 


19. april 2010.; 

Prihvaćeno: 

07. septembar 2010. 

SAŽETAK 


Cilj: Utvrditi uticaj biometeoroloških faza na incidencu suicida s 

obzirom na dob, pol, mesto stanovanja, dane u nedelji, mesece u 

godini, te način izvršenja suicida. 

Metode: Komparativna analiza podataka ispitanika o suicidima (s 

obzirom na period, mesece i dane u nedelji, mesto stanovanja, dob, 

pol, prosečnu starost) dobijenih iz Policijske uprave Kragujevac za 

period 2004-2008. godine i svakodnevnih biometeoroloških faza 

za grad Kragujevac dobijenih od Republičkog hidrometeorološkog 

zavoda (RHMZ) Srbije. 

Rezultati: U posmatranom periodu dogodila su se 144 suicida 

(14/100.000 stanovnika godišnje). Najviše suicida dogodilo se u 

2005. (36), a najmanje u 2007. (24) godini; najviše u januaru i 

junu (po 18), a najmanje u avgustu (7). Tri četvrtine slučajeva bili 

su iz urbanog okruženja. Tri četvrtine slučajeva su bili muškarci i 

to najviše u dobnim grupama 41-45 i 51-55 godina (po 11), a jedna 

četvrtina slučajeva bile su žene, najviše u starosnoj grupi 61-65 

godina života (7). Ukupna je prosečna starost iznosila 54,66 godina. 

Dve trećine svih suicida izvršeni su vešanjem (93) (od čega su 

dve trećine bili muškarci), a jedna šestina vatrenim oružjem (88% 

su bili muškarci). Najviše suicida dogodilo se u biometeorološkim 

fazama 4 i 9 (po 38), što čini više od polovine svih suicida. 

Zaključak: Statističkim metodama dobijena je značajna korelacija 

između biometeoroloških faza i incidence suicida, a najviše se 

suicida desilo u fazama stabilnog sunčanog vremena i naglog prodora 

frontova, s naglom promenom vremenske situacije. Preporuka 

ovog rada jeste striktno poštovanje upozorenja iz svakodnevne 

bioprognoze koju daje RHMZ Srbije, pa u ovim vremenskim prilikama 

posebnu pažnju treba obratiti na sve osobe sa suicidalnim 

tendencijama. 

Ključne reči: incidenca suicida, biometeorološke faze, vremenske 

prilike

UVOD 

Samoubistvo (suicidium) je svesno i namerno 

oduzimanje vlastitog života (1). Da bi neka osoba 

izvršila samoubistvo potrebno je da kod nje 

postoji suicidogena dispozicija i motiv (1). Suicidogena 

dispozicija ili suicidogeni mentalitet 

se opisuje kao prirodni ili stečeni manjak životnog 

nagona, te pojačane psihičke reaktivnosti na 

spoljašnje i unutrašnje sadržaje (1). Suicidogeni 

motiv predstavlja upravo onaj problem koji u nekom 

trenutku pobuđuje određenu osobu da učini 

samoubistvo (1). Suicidogeni impulsi često potpomažu 

suicidogene motive, a to su stanja koja 

olakšavaju donošenje odluke o samoubistvu, kao 

na primer, pubertet, menstruacija, klimakterijum, 

a po nekima i alkoholisanost, nedostatak droge 

kod narkomana i slično (2-6). 

Već je odavno uočeno da postoji određen odnos 

između samoubistva i vremenskih prilika, te je 

ustanovljeno da se samoubistvo pojavljuje u 

30-40% dana u godini i da određene vremenske 

prilike (jedan dan pre prolaza hladnog fronta ili 

dva dana nakon prolaza toplog) povećavaju broj 

samoubistava (7). Karakteristike tih perioda jesu 

nešto povišene temperature vazduha, nepravilnog 

dnevnog hoda, najčešće snižen ili promenljiv 

pritisak vazduha, moguća oblačnost, magla, 

često slaba kiša ili pred hladni front pljuskovi 

kiše, a u toplom delu godine neretko grmljavina 

ili sevanje. Ako frontalni poremećaji traju danima 

onda se i broj samoubistava znatno povećava 

u tim razdobljima (7) 

Prema američkom Nacionalnom institutu za mentalno 

zdravlje (National institute of Mental Health) 

najčešći faktori rizika za samoubistvo su: depresija 

i drugi mentalni poremećaji ili poremećaji 

zloupotrebe alkohola i narkotika (oko 90% svih 

pacijenata pripada ovoj grupi), prethodni pokušaj 

samoubistva, prethodna porodična istorija mentalnih 

bolesti ili zloupotrebe alkohola ili narkotika, 

porodična istorija samoubistava, istorija nasilja u 

porodici (uključujući fizičko ili seksualno nasilje), 

posedovanje oružja u kući (preko polovine svih 

samoubistava prema načinu izvršenja) i boravak 

i uticaj okoline koja podržava ili veliča samoubistvo, 

naročito članovi porodice, prijatelji, medijske 

ličnosti (22). Suicid je u 2006. godini bio sedmi na 

listi uzroka smrtnosti u svetu za muškarce i šesnaesti 

za žene, od toga je bio treći uzrok smrtnosti za 

osobe starosti 15-24 godine (8). 

Gajić et al Biometeorološki uticaj na suicid 

Uticaj vremena i klime na čoveka (biometeorologija) 

ogleda se kroz niz simptoma i bolesti (9, 

10). Biometeorološke prilike su izražene kroz deset 

biometeoroloških faza (10), a reakcije meteoropata, 

hroničnih bolesnika, posebno osetljivih 

ljudi, kao i ljudi sklonih suicidu, srazmerne su 

intenzitetu i promenama pojedinih meteoroloških 

parametara i vremenskih situacija (10). Pri relativno 

stabilnom vremenu reakcije meteoropata su 

smanjene ili ih nema (10). 

Zbog sve većeg broja samoubistava u savremenom 

svetu suicidologija je postala posebna tema 

multidisciplinarnog naučnog istraživanja (11). 

Učestalost, povećanje i smanjenje broja samoubistava 

u nekoj zemlji, zavisi od čitavog niza 

činilaca (11). S druge strane, i ranije je predmet 

mnogih studija bio pokušaj objašnjenja uticaja 

raznih, ali pojedinačnih meteoroloških i parameteoroloških 

faktora na pojavu suicida (12,13). 

Cilj našeg istraživanja bio je ispitivanje povezanosti 

uticaja vremenskih prilika na incidencu suicida 

njihovim objedinjavanjem u određene biometeorološke 

faze, te uporedna analiza i provera. 

ISPITANICI I METODE 

Metodologija našeg istraživanja zasnovana je na 

uporednoj analizi podataka o svakodnevnim biometeorološkim 

fazama za grad Kragujevac određenih 

na Odeljenju za primenjenu klimatologiju 

Republičkog hidrometeorološkog zavoda Srbije 

i podataka o suicidima dobijenih iz evidencije 

Odeljenja za seksualne i krvne delikte Policijske 

uprave Kragujevac za petogodišnji period 2004- 

2008. godine. Populaciju grada Kragujevca čini 

202.000 stanovnika, i to 100.000 muškaraca i 

102.000 žena. Uvidom u podatke o ispitanicima 

pratili smo sledeće parametre: distribuciju ispitanika 

po godinama (uzimajući svaku godinu posebno), 

po mesecima i zbirno za sve posmatrane 

godine, dane u nedelji i zbirno za sve posmatrane 

godine, mesto stanovanja, pol ispitanika, dobne 

grupe od pet godina (počev od 15. do 90. godine 

starosti), te prosečnu starost ispitanika prema posmatranim 

periodima ispitivanja. 

Biometeorološke faze, definisane u Republičkom 

hidrometeorološkom zavodu Srbije, jesu: CTS 

(ciklon, toplo, suvo), CTV (ciklon, toplo, vlažno), 

CTF (topli front), CHF (hladni front), CHV 

(ciklon, hladno, vlažno), CHS (ciklon, hladno, 

suvo), AHS (anticiklon, hladno, suvo), AHV (an- 

25

26 


Grafikon 1. Ukupan broj suicida 

ticiklon, hladno, vlažno), ATS (anticiklon, toplo, 

suvo) i ATV (anticiklon, toplo, vlažno) (14-19). 

Definicije samopovređivanja po dijagnozama definisane 

su u 10. međunarodnoj klasifikaciji bolesti, 

i to od X61 do X84 (20). 

Za statističku obradu podataka korištene su objektivne 

matematičko-statističke metode, adekvatno 

primenjene vrsti i tipu podataka i statističkom 

testu: metode deskriptivne statistike (tabeliranje, 

grafičko prikazivanje, apsolutne i relativne frekvencije) 

i metode neparametarskih statističkih 

testova (χ2 –test, studentov „t“ test i jednofaktorska 

ANOVA). Pritom verovatnoća p

Tabela 2. Načini izvršenja suicida s obzirom na pol 

Načini izvršenja / Execution ways 

Najviše je suicida izvršeno vešanjem (93) (dve trećine 

svih suicida), zatim vatrenim oružjem (25), te 

skokovima s visine (11) (p0,05) (Tabela 2). 

U biometeorološkim fazama 4 i 9 dogodilo se po 

38 suicida u svakoj (što zajedno čini polovinu svih 

akcidenata), u fazi 5 se dogodio 21 suicid (jedna 

šestina suicida), dok se u fazi 8 dogodio samo jedan 

slučaj za 5 godina (p0,05) (Grafikon 7). 

DISKUSIJA 

Muškarci 

/ Men 

Žene / 

Women 

Vešanje / Hanging (X70) 66 27 

Vatreno oružje / Firearms (X72-X74) 22 3 

Skok s visine / Jumping (X80) 7 4 

Samotrovanje / Selfpoisoning (X61-X69) 2 3 

Hladno oružje / Cold weapon (X78) 3 0 

Utapanje / Submersion (X71) 3 1 

Samozapaljenje / Selfcombusting (X76) 1 1 

Strujni udar / Electrocution (X83) 1 0 

Prema podacima Nacionalnog instituta za mentalno 

zdravlje SAD-a u 2009. godini na svaki 

uspešan suicid desilo se 12-25 pokušaja (21). 

Prema podacima Svetske zdravstvene organizacije 

iz 2008. godine, na prva četiri mesta po 

broju izvršenih samoubistava nalaze se zemlje 

bivšeg Sovjetskog Saveza, među kojima je prva 

Grafikon 4. Distribucija muškaraca i žena po dobnim grupama 


Grafikon 5. Načini izvršenja suicida 

Grafikon 7. Deo biometeoroloških faza sa suicidima u ukupnom 

broju BM faza 

Litvanija s incidencom 30,7/100.000 i Rusija s 

30,1/100.000 stanovnika (22). Među evropskim 

zemljama s najnižom incidencom su Grčka, Turska 

i Albanija (2,20; 3,94; 4,00/100.000). Među 

zemljama bivše Jugoslavije na prvom mestu je 

Slovenija, koja je ujedno i dvanaesta u svetu s 

19,8/100.000, potom Srbija, na trinaestom mestu 

u svetu s 19,5/100.000, Hrvatska, šesnaesta 

u svetu s 18,0/100.000, dok je Bosna i Hercegovina 

tek na 36. mestu s 11,8/100.000. U odnosu 

na navedene podatke incidenca od 14,4/100.000 

na lokalnom nivou grada Kragujevca, koju su 

Grafikon 6. Samoubistva prema biometeorološkim fazama 

27

28 


pokazali rezultati ovoga istraživanja, niža je od 

stope na nacionalnom nivou Republike Srbije. U 

gotovo svim državama veći je broj muškaraca u 

odnosu na žene, i to od 2:1 (Švajcarska, Hong 

Kong, Južna Koreja) (23,24,25) do 6:1 (Belorusija, 

Litvanija, Rusija, Poljska, Ukrajina) (22). 

Odnos među polovima dobijen iz rezultata ovoga 

istraživanja (2,69:1) sličan je nacionalnom nivou 

za Republiku Srbiju (2,56:1 za muškarce). 

Odnos muškaraca i žena od 4:1 u starosnoj grupi 

15-19 godina, te 6:1 u starosnoj grupi 20-24 

godine, zabilježen je u nekim istraživanjima (26, 

27, 28, 29). U našoj studiji taj odnos je 3:1 u obe 

navedene grupe, dok je u ostalim starosnim grupama 

od 2:1 do 5:1. Od ove distribucije odudara 

starosna grupa 61-65 godina u kojoj čak ima više 

žena od muškaraca (odnos 6:7) i starosna grupa 

76-80 godina gde je odnos 8:1 za muškarce. 

Nažalost, dobne grupe ispod 55. godine života u 

našem istraživanju činile su preko polovine svih 

suicida, što se poklapa s nalazima drugih istraživača 

(26-29). 

Prema nekim podacima najveći broj samoubistava 

se izvrši vatrenim oružjem (6 muškaraca na 4 

žene), vešanjem (2,2 muškarca na 2 žene) i trovanjem 

(1 muškarac na 4 žene) (26-29). U našoj 

studiji na prvom su mestu suicidi vešanjem (2,44 

:1 za muškarce), zatim vatrenim oružjem (8:1), 

dok je trovanje na četvrtom mestu (2:3), a žene u 

ovoj kategoriji nisu u velikoj prednosti kao u drugim 

istraživanjima (22). Ove se diskrepance pre 

svega objašnjavaju lakšim dobijanjem dozvole 

za nošenje oružja u SAD-u, zatim kulturološkim 

razlikama ove dve zemlje i uobičajenim, odnosno 

tradicionalnim načinima vršenja suicida u ovim 

sredinama (22). 

Tokom ovog istraživanja postojanje psihijatrijskog 

oboljenja nije uzimano kao poseban parametar 

jer biometeorološke faze imaju svoj uticaj 

na celokupnu populaciju, bez obzira na psihijatrijsku 

predistoriju. 

U nekim studijama je ustanovljena korelacija mesečnog 

ritma suicida s povišenim temperaturama, 

porastom trajanja dnevne svetlosti, povećanjem 

broja sunčanih sati ili padom vlažnosti vazduha 

(26-29), a što je potvrđeno i u našem istraživanju 

budući da se najveći broj suicida desio u 

fazama 4 i 9 koje karakteriziraju gore navedene 

vremenske prilike. Negativna korelacija između 

ukupnog mesečnog broja suicida i broja sunčanih 

sati (biometeorološka faza 9 u našem istraživanju) 

takođe je ustanovljena u prethodnim istraživanjima 

(30-32). Uticaj sezonaliteta vremenskih 

prilika ustanovljen je poređenjem broja suicida s 

frekvencijom pojave pojedinih biometeoroloških 

faza tokom kalendarske godine, budući da se biometeorološka 

faza 9 najčešće javlja u prolećnim 

i letnjim mesecima, dok je faza 4 prisutna u svim 

godišnjim dobima, ali je njen uticaj najizraženiji 

takođe u prolećno-letnjim mesecima (33-35). 

Kako se formiranjem hladne vazdušne mase u samoj 

zoni nadolazećeg fronta javlja vertikalni razvoj 

oblačnosti, to ispred ovog polja naglo povećava 

koncentraciju pozitivnih jona koji izuzetno 

negativno utiču na raspoloženje i zdravlje ljudi 

(33-35). Tako se javlja pad koncentracije, uznemirenost, 

osećaj teskobe, gušenja, pad kognitivnih 

i voljnih funkcija, što konsekutivno vodi ka 

depresiji i lošem raspoloženju. Kako je depresija 

i loše raspoloženje izuzetno dobra podloga za suicidalne 

ideje, to je i frekvencija suicida u ovoj 

fazi izraženija (28,29). Pozitivni joni se javljaju 

kod frontalnih kretanja vazduha i do 24 sata pre 

nevremena. Hronični bolesnici tada osećaju niz 

nelagodnosti i pogoršanje zdravstvenog stanja, te 

mogu unapred da osete promenu vremena (37). 

S porastom koncentracije negativnih jona raste 

osećaj prijatnosti i zadovoljstva, te se povećava 

sposobnost koncentracije. Dok se u naseljenim, 

urbanim oblastima oni lako apsorbuju vezujući 

se za aerozagađivače, metalne konstrukcije, klima-uređaje, 

radijatore, pa im na taj način koncentracija 

opada, najviše su koncentracije prisutne 

pored vodopada, u planinskim i šumovitim 

predelima (37). 

Rezultati ovoga istraživanja pokazali su značajnu 

korelaciju između pojedinih biometeoroloških 

faza i incidence suicida, što je i bio cilj našeg 

istraživanja. Najviše se suicida dogodilo u biometeorološkoj 

fazi 9, odnosno za vreme stabilnog 

sunčanog vremena i tokom naglog prodora 

frontova, tokom biometeorološke faze 4, kada se 

vremenska situacija naglo menjala, najčešće sa 

suvog toplog na hladno vlažno vreme. Kako su 

ovo ujedno i faze s najvećom frekvencijom pojavljivanja 

u toku godine, tako je u ovim vremenskim 

prilikama potrebno posebnu pažnju obratiti 

na sve osobe sa suicidogenim mentalitetom i 

suicidogenim impulsima. Zaključak ovog istraživanja 

nalaže poštovanje upozorenja iz svakod-

nevne bioprognoze koju daju regionalni i lokalni 

hidrometeorološki zavodi, što znači redovno 

uzimanje ranije propisane terapije i redovne periodične 

kontrole psihijatrijskih bolesnika. Osim 

toga, potrebno je osnovati posebne SOS ili „call“ 

centre koji bi reagovali u kriznim situacijama i u 

svim slučajevima kod kojih postoje suicidogene 

tendencije i suicidogeni motivi. Kod pacijenata 

s hroničnim psihijatrijskim poremećajima modus 

bi se trebao tražiti u posebnim službama koje bi 

se bavile povremenim kućnim posetama uz va- 

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lidaciju psihijatrijskog statusa, naročito u situacijama 

kada se najavljuju učestale meteorološke 

pojave, tipa biometeorološke faze 4 ili 9. U ovakvim 

vremenskim prilikama naročitu pažnju treba 

obratiti na tzv. samoubice povratnike, odnosno 

one koji su za sobom već imali neki neuspeo 

pokušaj suicida. 

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klasifikacija bolesti. (internet izdanje) (maj 2009) 

http://www.stetoskop.info/mkb.php 

21. Centers for Disease Control and Prevention. 

National 

Center for Injury Prevention and Control. Webbased 

Injury Statistics Query and Reporting System 

(WISQARS) : www.cdc.gov/ncipc/wisqars (maj 

2009.) 

22. Gibbons RD, Brown CH, Hur K, Marcus SM, Bhaumik 

DK, Erkens JA, Herings RM, Mann JJ. Early 

evidence on the effects of regulators’ suicidality warnings 

on SSRI prescriptions and suicide in children 

and adolescents. Am J Psychiatry 2007; 164:1356- 

63. 

23. Salib E, Gray N. Weather conditions and fatal selfharm 

in North Cheshire 1989-1993. Br J Psychiatry 

1997; 171:473-7. 

24. Kok LP, Tsoi WF, Fung M. The wish to die: suicidal 

behaviour in Singapore. Singapore: Samaritans of 

Singapore, 1993. 

25. Kalkstein LS, Davis RE. Weather and human mortality: 

an evaluation of demographic and interregional 

responses in the United States. Ann Ass Am Geographers 

1989; 79:44-64. 

26. Parker G, Gao F, Machin D. Seasonality of suicide 

in Singapore: data from the equator Psychol Med 

2001; 31:549-53. 

29

30 


27. Parker G, Walter S.S. Seasonal variation in depressive 

disorders and suicidal deaths in new South Wales. 

Br J Psychiat 1982; 140: 626–32. 

28. Miyake S, Hashimoto M, Iwashita J, Suzuki K, 

Kitano M. Effects of negative air ions on task performance, 

mood and physiological indices. U: Proceeding 

of 4th International Conference on Psychophysiology 

in Ergonomics, Glasgow Great Britain, 

22.september 2002. The University of Glasgow, 

Glasgow Great Britain, 2002. 

29. Hakko H. Seasonal variation of suicides and homicides 

in Finland. Department of Psychiatry University 

of Oulu, Department of Forensic Psychiatry University 

of Kuopio, 2000, disertation 

30. Preti A, Miotto P. Seasonality in suicides, the influence 

of suicide method, gender and age on suicide 

distribution in Italy. Psychiat Res 1998; 81:219–31. 

31. Eisenbach C, Ungur Al, Unger J, Stremmel W, Encke 

J. Admission to intensive care for parasuicide by 

self-poisoning: variation by time cycles, climate and 

the lunar cycle. Psychiatry Res 2008; 161:177-84. 

Influence of biometeorological phases on incidence of suicides 

32. Zung WWK, Green RL. Seasonal Variation of Suicide 

and Depression. Arch Gen Psychiatry 1974; 

30:89-91. 

33. Tiihonen J, Rasanen P, Hakko. H. Seasonal variation 

in the occurrence of homicide in Finland. Am J Psychiat 

1997; 154:1711–4. 

34. Retamal P, Humphreys D. Occurrence of suicide 

and seasonal variation. Rev Saude Publica 1998; 

32:408–12. 

35. Chew KS, McCleary R. The spring peak in suicides: 

a cross-national analysis. Soc Sci Med 1995; 

40:223–30. 

36. Linkowski P, Martin F, De Maertelaer V. Effect of 

some climatic factors on violent and non-violent suicides 

in Belgium. J Affect Disord 1992; 25:161-6. 

37. Jevtić M, Rosić I, Jovašević LJ, Veljović M. Balneoklimatologija 

za ekonomiste. Kragujevac: Komino 

Trade, 2005, III:23-30. 

Vladimir Gajić¹, Dragan Milojević¹, Jasminka Smailagić², Nela \onović³, Suzana Matejić4 , Sanja Gajić 5 

¹Institute for Emergency Medical Care of Kragujevac, ²Republican Hydrometeorological Institution of Serbia, ³ Public Health Institute of 

Kragujevac, 4 School of Medicine of the University of Kragujevac, 5 Health Service of Kragujevac; Kragujevac, Serbia 

ABSTRACT 

Aim To establish an influence of biometeorological phases on suicide incidence according to age, gender, 

settlement, week days, months in a year, ways of suicide execution, influence of biometeorologic 

phases on suicide execution. 

Methods Comparative analysis of the data about suicides (by years, months, week days, settlements, 

age, gender) obtained by the Police Department in Kragujevac for the 2004-2008 period and everyday 

biometeorological phases (phase 1 CWD: cyclone, warm, dry; phase 2 CWW: cyclone, warm, wet; 

phase 3 CWF: cyclone, warm front; phase 4 CCF: cyclone cold front; phase 5 CCW: cyclone, cold, wet; 

phase 6 CCD: cyclone, cold, dry; phase 7 ACD: anticyclone, cold, dry; phase 8 ACW: anticyclone, cold, 

wet; phase 9 AWD: anticyclone, warm, dry; phase 10 AWW: anticyclone, warm, wet) in Kragujevac 

determinated by the Republic Hydrometeorology Institute of Serbia. 

Results In the observed period there were 144 suicides resulting in the incidence rate of 14/100 000/ 

year. The highest number of suicides was noted in 2005 (36), and lowest one in 2007 (24). The most 

suicide cases happened in January and June (18 in each), and the fewest in August (7). Three quarters 

occurred in urban areas. Three quarters of victims were males mostly in the age groups 41-45 and 51-55 

(11 in each). Females made one quarter of victims and most of them were in the age group 61-65 years 

(7). Total mean age was 54,66 years. Two thirds of all suicides were executed by hanging (93) (two 

thirds were among males), one sixth by firearms, with nine tenths of men. The most incidents with significant 

correlation were in biometheorological phases 4 and 9 (stable sunny weather and sudden impact 

of weather fronts, with sudden weather changes) (38 in each), which makes over a half of all suicides. 

Conclusion According to the results of this study, it is strongly recommended to monitor everyday 

biometeorological forecast , and special attention must be paid to all persons with suicide tendencies. 

Key words: suicide incidence, biometeorological phases, weather conditions 

Original submission: 11 November 2009; Revised submission: 19 April 2010; Accepted: 07 September 2010.


Colorectal cancer early detection program integrated in practice 

of family physicians 

Sanda Pribić, Ljiljana Trtica - Majnarić, Rudika Gmajnić, Marko Lukić, Nada Prlić 

Medical School Osijek, Osijek, Croatia 

Coresponding author: 

Rudika Gmajnić 

Health Centre Osijek 

Park Kralja Petra Krešimira IV 6 

Phone: +385 31 225 400; 

fax.: +389 31 225 330 

E-mail: rudika.gmajnic@os.t-com.hr 


26 July 2010; 


19 August 2010; 

Accepted: 

08 September 2010. 

ABSTRACT 


Aim To present preliminary results of the colorectal cancer early 

detection program, a part of the project called „A Model of Early 

Cancer Detection Integrated in a Practice of a Family Physician“, 

carried out by the Department of Family Medicine of the Osijek 

University School of Medicine and the Health Centre of Osijek, 

Croatia. 

Methods The strategy of the project, based on the central role of a 

family physician in the implementation of the early cancer detection 

programs, was described and preliminary results of the colorectal 

cancer early detection program are presented and compared 

with the same issues of the National Program, centrally conducted 

and supplied by public services. 

Results From the beginning of April unil the end of May 2009, a 

total number of 516 testing cards on occult faecal blood were delivered 

to patients from two target groups (aged 45-50 and 75-79). 

A high responding rate of 69,76% (360) was recorded. This is an 

advantage in comparison with the low responding rates of about 

20% (43 862), obtained by the National Program. In the project, 

there were in average 2,5% (9) positive tests, with the higher percent 

in the older than in the younger age group, 3,5% (12) and 1% 

(4) respectively). 

Conclusion Data obtained by the Project, and by the National Program 

- indicate that there could be a need for a more precise definition 

of risk groups who have to be invited for screening. 

Key words colorectal cancer, early detection, strategies, the National 

Program, integrated program 

31

32 


INTRODUCTION 

Colorectal cancer is currently on the second position 

by both incidence and mortality rate among 

malignant tumours in EU countries (1). It is one 

of the fastest growing types of cancer in western 

countries, which is supposed to be a consequence 

of modern lifestyle such as unhealthy diet, rich in 

saturated fat and poor in vegetables and fruits, as 

well as lack of body exercises (2). 

On the other hand, it has an excellent possibilities 

to be cut if recognized and cured at an early 

stage. Theoretically, all colon and rectal cancers 

can be either prevented or cured by removal of 

adenomas and cancers in their early stages (2,3). 

Past experience in using conventional screening 

method for early detection of the colorectal cancer, 

Fecal Occult Blood Test (FOBT), applied 

in asymptomatic population at average risk, has 

shown that 3-5% persons with positive tests could 

be expected (4,18). They are candidates for 

further evaluation by colonoscopy (4,18). Following 

up this protocol, it is possible to decline 

the mortality rates for 18-33% (4). 

In Croatia, both the colorectal cancer incidence 

and the mortality rates are on a steady increase 

(14). It is ranking second in prevalence for both 

sexes, following lung cancer in men and breast 

cancer in women (5). A need for urgent implementation 

of systematically performed screening 

on colorectal cancer on a national scale is 

illustrated by the fact that high mortality rates 

(varying between 2000 and 2006 from 37,5 to 

40,5/100,000) have been recorded (5). It can partially 

be a consequence of the fact that it is detected 

too late (6). 

In the Osijek-Baranja County, the increasing 

trend of the mortality rates for colorectal cancer 

even exceeds the average for Croatia. One of the 

reasons of such unfavourable statistics may be the 

war in 1991/92, the Osijek-Baranja County was 

faced with, and the long post-war period leaving 

the negative consequences on the local economy 

and health of the citizens (7,8). 

After several years of preparations and following 

the international recommendations, the National 

Program for Early Detection of Colorectal Cancer 

was implemented in November 2007. This 

program is a part of a more ambitious project for 

early detection of four main cancer sites proved 

to be preventable, including also breast, prostate 

and cervix uteri (9-12). 

Taking into account the unfavourable colorectal 

cancer statistics and using their own experiences 

in performing systematic screening on colorectal 

cancer, the leaders of the Department of Family 

Medicine of the Osijek University School of Medicine 

and the Health Center Osijek introduced 

at the same time the project ‘’A Model of Early 

Cancer Detection Integrated in Practice of Family 

Physicians’’ (13). An idea promoted by the 

project is that screening and early diagnosis of 

cancer is more efficient if integrated in practice 

of family physicians, compared to the National 

Program, centrally directed and supplied by the 

public services. 

This paper presents preliminary results of the colorectal 

cancer early detection program obtained 

by the project. Possible advantages of this model 

are emphasized as compared to the alternative 

model proposed by the National Program. 

RESEARCH SAMPLE AND METHODS 

Croatian National Program of Prevention and 

Early Detection of Colorectal Cancer 

The main objectives of the project was to achieve 

the screening coverage of at least 60%, to reverse 

the low proportion of diagnosed pre-clinical 

and localised cancers, to reduce mortality by 

15% during the period of five years after the program 

started. Specific goals are directed towards 

improvements in diagnostics and treatment and 

standardization of protocols (10-12). 

The target population was defined according to 

international recommendations and include males 

and females aged 50-74 at average risk (10- 

12). 

The screening protocol was based on two-year 

checkups with the FOBT. Persons with positive 

tests are referred to colonoscopy in the nearest endoscopic 

unit (10-12). Based on widely obtained 

data, 10-15% of them are expected to be diagnosed 

colorectal cancer and 30-40% adenomas (3). 

Special protocols including the beginning of the 

screening much earlier in the life and more frequent 

colonoscopic examination are planned for population 

groups at higher than average risk, such 

as patients with inflammatory bowl diseases, or

persons with a positive family history of colorectal 

cancer and polyposis syndromes,. 

The Croatian Public Health Institute coordinates 

and monitors the activity, also taking care of 

arrangement of invitations, collection and evaluation 

of data (10-12). 

Specialists colonoscopists, surgeons, as well as 

laboratory teams and coordinators employed 

in the counties` Public Health Institutes are included 

in the program implementation. Family 

physicians are not formally included, only when 

they refer for follow up treatment those patients 

whose diagnosis of cancer was confirmed by colonoscopy 

(10-12). 

Invitation letters are sent by mail to home addresses. 

An envelope contains three testing-cards, 

instructions for their use, questionnaire about risk 

factors and an educational brochure to ensure that 

invited persons are informed on screening. Invited 

persons are asked to mail testing-cards back, 

after they have used them, together with a filled 

questionnaire. The purpose of the questionnaire 

is to obtain information on risk factors from the 

invited persons, including: nutrition, consumption 

of alcohol, low physical activity, overweight, 

history of colon diseases and family history of 

colorectal tumorous diseases. This is expected to 

allow insight into the distribution of risk factors 

in the target population (10-12). 

The project - „A Model of Early Cancer Detection 

Integrated in Practice of Family Physicians“ 

The project started in March 2007 (13). Early detection 

protocols for all four preventable cancer 

sites, including breast, colon and rectum, cervix 

uteri and prostate are planned to be implemented 

by the project . 

Objectives of this trial were to assess the acceptability 

and applicability of an early cancer detection 

based on active and systematic approach of a 

family doctor (13). 

At the beginning, the project was designed as 

the „pure“ integrated model. This means that a 

family physician takes full responsibility for all 

stages of the screening protocol, including invitation 

arrangements and encouragement of patients 

to participate in the research (13). 

As the preparations for the implementation of 

the programs for early detection of breast and 

Pribić et al Cancer detection program in family medicine 

colorectal cancer went in parallel with those in 

the National Program, some modifications of 

planned protocols of colorectal and breast cancer 

were done. The main objectives of the project 

included questions expected to be answered by 

the four-year research: 

Can the coverage of the target population be significantly 

increased as compared to the alternative 

approach introduced by the National Program? 

Are family physicians sufficiently educated and 

motivated for the program implementation and 

how can this be improved if necessary? 

Personnel, time, equipment and costs needed 

How can better interdisciplinary collaboration in 

performing preventive programs be achieved? 

How can the program be implemented to be tailored 

to the present facilities and resources? 

Colorectal cancer Early Detection Program 

The screening program on colorectal cancer started 

first in April 2009, after two years of preparations. 

Twenty GP teams in the Osijek region, both 

from urban and rural areas, who gave their informed 

consents, participated in the research. All 

family physicians who were included are respective 

professionals. Several of them are currently 

attending the vocational training and the others 

are specialists. 

In order to avoid the interruption of the National 

Program implementation, the definition of the target 

population was changed first. Thus, subjects 

included in the program belong to either the 5-year 

lower class (45-49), or the 5-year upper class 

(75-79) as compared to the official target population 

defined by the National Program. These groups 

encompass approximately 4,000 people, randomly 

selected from a large sample of the total of 

27,000 subjects recorded on the lists of 20 family 

physicians included in the project. The same age 

groups recorded on the lists of family medicine 

teams, who are not included in the project were 

used as control groups. 

All necessary preparations for the project implementation 

were done during first two years of research 

(16). Primarily, the task was to select the 

target population for all four cancer sites planned 

to be investigated. The defined target groups were 

informed on measures of the primary prevention, 

risk factors, early signs of a malignant disease 

33

34 


and methods for early cancer detection. For this 

purpose, educational courses and lectures were 

held in the local community units and the Health 

Centre of Osijek. In collaboration with the City 

of Osijek, League Against Cancer, health education 

brochures and posters were issued. Citizens 

and people from the target groups can also get 

information on cancer-related issues on the web 

site www.prevencijaraka.com. 

Physicians included in the project were interviewed 

to obtain their opinion about the usefulness 

of the early cancer detection program implementation 

in their regular practice and to assess their 

motivation to participate in the research. 

The computer program 

Computer sofrware for keeping the records on data 

and follow up on the research protocol was designed 

and installed for the application in GP offices 

included in the project. Data on patients selected 

from the target population are downloaded from 

Table 1. New colorectal cancer cases diagnosed per year in 

Croatia* 

YEARS 

Colon, rectum, 

sigma et 

anus 


sigma et 

anus 


sigma et 

anus 

M F TOTAL 

1976 17,2 20,2 18,8 

1977 21,7 20,4 21,1 

1978 23,6 20,1 21,8 

1979 26,8 22,8 24,7 

1980 26,1 22,1 24,0 

1981 26,1 24,0 25,0 

1982 27,8 24,5 26,0 

1983 26,0 25,4 25,8 

1984 28,9 25,6 27,2 

1985 31,0 30,7 30,8 

1986 33,8 26,7 30,1 

1987 28,4 28,0 28,1 

1988 33,9 28,4 31,1 

1989 32,2 31,9 32.0 

1990 36,1 32,9 34.5 

1991 33,7 29,8 31.7 

1992 38,2 31,1 34.5 

1993 37,8 30,6 34,1 

1994 40,8 33,5 37,1 

1995 47,3 33,4 40,1 

1996 46,4 33,4 39,7 

1997 50,5 39,1 44,6 

1998 52,9 41,0 46,8 

1999 71,3 53,0 61,7 

2000 73,0 54,0 63,1 

2001 65,2 47,6 56,1 

2002 72,2 48,7 60,1 

2003 70,1 53,6 61,6 

2004 71,7 48,7 59,8 

2005 75,7 53,3 64,0 

2006 74,6 51,2 62,4 

*Source: Cancer register, Croatian Public Health Institute (14) 

the CEZIH system (central informational network 

providing support to the Health Care System), 

which contains records of all insured citizens. 

The software was designed to select patients 

according to their sex and age in one or more target 

groups. Continuity of the screening process 

(by two-year examination protocol) is ensured in 

the way that a patient is retained on the list of 

the program until the physician certifies with his/ 

her signature that the diagnostic procedure for 

this particular patient is completed. The record of 

that patient can be downloaded from the CEZIH 

system again only after a specifically defined period 

of time (Figure 1). 

There are separate menus for each of four cancer 

sites under investigation. Except personal data, a 

menu contains data on diagnostic and treatment 

procedures, as well as cancerous lesion-related 

data, including localisation, spreading and pathohistologic 

diagnosis for surgically treated patients 

(Figure 1). Date and main cause of death can also 

be recorded as important for the estimate of the 

5-year survival rate and cancer-related mortality. 

Colorectal cancer early detection protocol 

Family physicians call their patients from the 

target groups by phone, deliver them letters of 

invitation in envelops together with three testing 

cards, brochure for their use and a questionnaire 

on risk factors. The physician also provides instructions 

on how to correctly apply the testing 

cards and all other issues the patients may be interested 

in. A physician reads the applied testing 

cards when patients return them back and makes 

the record on the results. Patients with positive 

tests are referred for further diagnostics by colonoscopy. 

The physician keeps records on all data 

fields included in the software. 

Figure 1. Computer program for keeping records on data

RESULTS 

Persons aged 50-74 were covered first by the National 

Program. The reason why these advanced 

age groups were invited first is the statistics indicating 

the highest incidence of colorectal cancer 

in older age (11). 

Until March 2009 the total number of 206,168 

invitation letters had been sent for these age groups 

within the National Program (Table 2) (14). 

Early responding rates, based on estimate of the 

total number of envelopes mailed back, were low 

(21,3% in average) (Table 2) (14). Tests inaccurately 

applied or unapplied were excluded as the 

results would have been even worse. 

In general, the results are the worst for the oldest 

population groups born in 1933 and 1934, and for 

the first year of screening. 

The percentage of the FOBT positive tests in the 

Osijek-Baranja County is 11,88%. The number 

of newly diagnosed cancer cases until now has 

been 22. 

Results of the colorectal cancer early detection 

program integrated in practice of family physicians 

covering the priod until May 31st 2009 show 

the total number of 516 cards testing occult faecal 

blood were delivered to all 516 patients. 69,76% 

(360) of patients have returned applied tests back 

Table 2. Preliminary results of the Croatian national colorectal 

cancer early detection program * 

For subjects born in 

1933, 1934, 1936, 1937 No. % 

envelopes mailed (of totally planned) 206,168 

envelopes mailed back 43,862 21,3 

applied tests (responding rate) 36,959 17,9 

inaccurately applied tests 1453 0,7 

responding rate after non-applied and inaccurately 

applied tests are excluded 

examined tests 35,684 

17,2 

positive tests 3,385 9,5 

the colonoscopic examination results 

invited for colonoscopy 2680 

not responded 416 

examinations done 1829 

negative results 317 

positive results 

diagnosis confirmed 

1425 77,9 

carcinoma 120 

polypus (adenomas) 645 

hemoroidal disease 371 

diverticulosis 264 

other reasons of occult faecal bleeding 

faecal bleeding 

74 

*Source: Cancer register, Croatian Public Health Institute (14) 


(Table 3). There were in average 2.5% positive 

tests, predominantly from people of older age group 

(aged 75-79). Only two cases from the younger 

target group (aged 45-49) had positive tests (Table 

3). One of them has the positive family history on 

polyposis syndrome and the other had suffered for 

a long time from the colon expulsion inability. 

DISCUSSION 

Data analysed so far have clearly shown that a 

response to colorectal cancer screening was much 

better when it is organized under the responsibility 

of family doctors than when it is conducted 

completely by public institutions (responding rate 

is approximately 70% and 20%, respectively) 

The results are comparable to those obtained by 

the first population-based study on colorectal 

cancer screening performed in the Osijek-Baranja 

County as early as three decades ago (17). 

The study was conducted on a large representative 

sample of 9259 asymptomatic people over 40 

and older. They were invited for screening by the 

letters mailed to their home addresses. After two 

years of research the results of the study confirmed 

the occult faecal blood testing as the standard 

method for early detection of colon carcinoma, 

because of the good proportion of sensitivity 

and the specificity of the test (sensitivity 72,22%, 

specificity 99,07%) (18). In addition, it was proved 

to be simple and safe for use and acceptable 

for the population (17). In comparison with the 

costs needed to cure patients in advanced stages 

of the disease, colon cancer screening proved to 

be an economically justified procedure. Based on 

these results, local health authorities, responsible 

for the study performance, have initiated numerous 

activities to improve cancer early detection 

and cancer care (19-21). The aim was to increase 

awareness among physicians and the public that 

cancer is preventable and that by detecting and 

curing it in its pre-cancer or early cancer stage it 

is possible to increase the mean life expectancy 

of cancer patients (22,23). 

Table 3. Colorectal cancer early detection program integrated 

in practice of family physicians, early results 

Delivered 

tests 

Returned 

tests 

Inaccurately 

applied tests 

Positive 

tests 

No. % No. % No. % No. % 

45 - 49 198 38,3 144 72,7 6 3 2 1 

75 - 79 318 61,6 216 67,9 19 6 11 3,5 

Total 516 360 69,76 25 4,84 13 2,5 

35

36 


Based on these long-term experiences in performing 

cancer-related programs in the local 

community, local health authorities assume that 

cancer early detection measures have to be focused 

on the Primary Health Care services, with 

the central role of family physicians (16). A simulated 

model of early detection of colorectal 

cancer was performed in order to estimate time 

and costs required. In general, the model showed 

that no more than 2 or 3 patients should be added 

per day, consuming as much as 20-50 minutes 

of an extra time of a family medicine team, depending 

on whether it is the first examination or 

a follow-up of cases with positive results. This 

was an argument that it is possible to integrate 

the colorectal cancer early detection program 

into the official health care system, without the 

need for substantial organizational changes and 

additional finance. The project is based on active 

and systematic approach of a family doctor. 

That means that the doctor actively performs 

preventive measures, recognizing medical needs 

of the patients and following the guidelines. This 

approach is different from that whereby the doctor 

generally responds to patient’s requests (15). 

Family physicians possess excellent possibilities 

to perform preventive activities, because of their 

specific position in the health care system, in the 

front line contact with the population (24). Their 

attitudes are that the prevention and early detection 

of cancer is necessary and still insufficiently 

implemented in practice. Moreover, preventive 

activities should become a centre of their occupation. 

In order to implement these programs within 

routine practice, they suggest certain organizational 

prerequisites. The emphasis is placed 

on the decrease of the number of patients on their 

lists and a more precise definition of the working 

frame of family medicine as a specific medical 

profession. Family physicians included in the 

project were additionally educated on cancer early 

detection methods. 

When data concerning positive tests are analysed, 

some important observations can be made. 

In general, it seems that older part of the conventionally 

defined target group included in the 

National Program and already covered by screening 

(aged 70-74), are preferentially disposed 

to colorectal occult bleeding and even for the 

development of cancer (11,88% positive results 

for the Osijek-Baranja County). This could be in 

accordance with the statistics showing that colorectal 

cancer incidence in Croatia, in the groups 

aged 60 years and more, largely exceeds the younger 

groups (78,3% compared to 7,9% in the 

groups up to 50 years of age) (5). High rates of 

positive tests obtained by the National Program 

can partially be a consequence of a long period of 

absence of systematically performed screening. 

Data obtained by the project indicate that in the 

middle age population groups (45-49) a very low 

rate of positive tests on systematically and non 

selectively performed screening can be expected 

(1% positive tests). This indicates that, for younger 

population groups, information on family history 

of colorectal cancer and other colon related 

diseases, as well as colon related symptoms and 

signs, should be obtained first in order to select 

persons at higher than average risk for the development 

of colon cancer. 

Nevertheless, based on the results of this study 

screening of colorectal cancer using hemoccult 

test proved to be an economically justified procedure 

(17). There is a growing awareness worldwide 

that only global action can make a dramatic 

stride in taking the control over the spreading of 

malignant disease (25-27). The main objective set 

by international bodies is to increase the number 

of countries that have the national cancer control 

program, covering cancer prevention, early detection, 

treatment, palliative care and support to 

cancer patients (28-31). This is equally important 

for the developing and the developed countries, 

as it is estimated that more than 70% of all cancer 

deaths occur in low income countries. Bearing in 

mind the differences in socioeconomic, cultural 

and resource settings among countries, the world 

health authorities have stated that all countries 

should adopt evidence-based guidelines and quality-assured 

national programs for early detection 

and treatment of cancer, but based on their own 

infrastructure (26). World experiences gained so 

far in the implementation of national programs 

on colorectal cancer early detection show that 

the best results are achieved when programs are 

performed in a centralized and standardized fashion 

(2). This includes a central agency with an 

established call/recall system to ensure equality 

in access to screening for all people from the 

target population, evaluation of tests in a central

laboratory facility and a centrally compiled and 

evaluated data. The main problem in all national 

programs is how to increase the screening rates. 

Experiences from Finland, where the best participation 

rate of about 70% has been achieved, 

clearly indicates that health-care services, when 

arranged primarily at the local level, as well as 

a strong public-oriented health-care system, may 

provide a substantially positive impact on early 

cancer detection programs implementation (2). A 

central role of the family physician in the implementation 

of preventive programs has been recognized 

as an advantage in terms of achieving 

better screening coverage and decreasing costs 

as compared to the strictly centrally controlled 

programs (32). However, even those who advocate 

for this approach point out that some kind 

of technical and professional support to family 

physicians has to be ensured, e.g. establishing a 

reminder system, standardizing the preventive 

policy, or encouraging physicians to use evidence-based 

guidelines and improve doctor-patient 

communication skills (33). 

In Great Britain, family physicians actively participate 

in the implementation of preventive programs. 

This has led to the quality indicators development 

which serve as the basis for the payment, 

in the form of an additional contract concluded 

with the Health Insurance Institutes (34). 

In general, there is no doubt that screening of colorectal 

cancer is effective, but there is no consensus 

about which screening strategy is more 

efficient than another in a real situation within 

the established health care system (35). 

Preliminary results obtained by the project show 

a high response rate compared to that obtained 

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2. IUCC/International Union against Cancer. Three year 

evaluation report. International Union against Cancer, 

2007. http://www.iucc.org/index.php?option=com 

content&task=view&id= 015983&Itemid= 

3. American Cancer Society. Five-year relative survival 

rates by stage at diagnosis 1996-2002. Cancer 

Facts and Figures. Bethesda:American Cancer Society; 

2007. 

4. Bond JH. The place of fecal occult blood test in colorectal 

cancer screening in 2006: the U.S. perspective. 

Am J Gastroenterol 2006; 101:219-21. 

5. Hrvatski zdravstveno-statistički ljetopis. Zagreb: 

Hrvatski zavod za javno zdravstvo, 2007. 


by the National Program. More positive tests, as 

expected, can be found in advanced age groups. 

Data obtained by the both strategies, one where 

family physicians take responsibility for the 

program implementation, and the other centrally 

directed and supplied completely by the public 

services, indicate that there could be a need for a 

more precise definition of risk groups to be invited 

for screening. 

However, it is too early to make final conclusions. 

We will be able to think about them more 

precisely in two years and more, when the project 

is closed and a larger pool of data is collected and 

evaluated. It is also expected that in the coming 

years a better insight into the National Program 

implementation efficiency and outcomes will be 

provided. 

ACKNOWLEDGMENTS/DISCLOSURES 

Population-based study, a part of the project titled 

‘’A Model of Early Cancer Detection Integrated 

in Practice of Family Physicians’’, carried 

out by the Department of Family Medicine of the 

Osijek University School of Medicine and the 

Health Centre of Osijek, No. 21-1061871-2087, 

sponsored by the Croatian Ministry of Science, 

Education and Sports. 

We appreciate the efforts of the general practitioners 

and patients who participated in the study. 

We wish to acknowledge the contribution of the 

Health Centre of Osijek, who have allowed the 

implementation of the project in the facilities of 

the Health Centre of Osijek. We also thank computer 

programmers who designed software to support 

data collection. 


6. 

7. 

8. 

Ebling Z, Hadžić N, Strnad T, Kolevska-Kaniški A, 

Kratković A. A fifteen-year survival in surgically 

treated patients with colorectal carcinoma. Croat J 

Gastroenterol Hepatol 1993; 2:125-30. 

Prlić L, Ebling Z, Glavina K, Gmajnić R, Vuletić G, 

Kovačić L. Health of returnees in Osijek Region and 

required special measures of health care and community 

organization. Coll Antropol 2004; 28 (suppl 

2):345-56. 

Ebling B, Trtica-Majnarić Lj, Gmajnić R, Ebling Z, 

Vranješ Z. Psycho-social aspects of measures aimed 

at decresing prevalence of chronic diseases in the 

population of returnees in the Osijek Region, Croatia. 

Coll Antropol 2007; 31:315-19. 

37

38 


9. Šamija M, Strnad M, Ebling Z. Prijedlog nacionalnog 

programa prevencije i ranog otkrivanja raka. 

In: Šamija M, Solarić M, editors. Zbornik radova 

2. kongresa Hrvatskog onkološkog društva, Opatija. 

2004. Zagreb:Hrvatsko onkološko društvo HLZ, 

2004: pp.:35-7. 

10. Šamija, M, Strnad M, Ebling Z, Kovačić L, Znaor 

A. In: Prijedlog Nacionalnog Programa prevencije 

i ranog otkrivanja raka u Hrvatskoj. Hrvatsko onkološko 

društvo, Ministarstvo zdravstva i socijalne 

skrbi, Hrvatski zavod za javno zdravstvo, Hrvatski 

zavod za zdravstveno osiguranje,ed. Zagreb: Art Design 

Šiško, 2006. 

11. Šamija, M, Strnad M, Ebling Z. How to prevent and 

detect cancer early? In: Draft National Program. 

Croatian Oncological Society, ed. Zagreb: Medicinska 

naklada, 2007. 

12. Ministarstvo zdravstva i socijalne skrb. Nacionalni 

program ranog otkrivanja raka debelog crijeva. Zagreb: 

Ministarstvo zdravstva i socijalne skrbi, 2007. 

13. Ebling Z. Znanstveni projekt ‘’Model ranog otkrivanja 

raka integriran u praksu obiteljske medicine’’ 

(Project No 21-1061871-2087) [Plan projekta] Osijek: 

Medicinski fakultet u Osijeku, 2007. 

14. Hrvatski zavod za javno zdravstvo Osijek. Registar 

za rak debelog crijeva. http://www.zzjzosijek.hr/ ( 

April 2009) 

15. Katić M, Jureša V, Orešković S. Family medicine in 

Croatia: past, present and forthcoming challenges. 

Croat Med J 2004; 45:543-49. 

16. Majnarić Trtica Lj, Strnad M, Gmajnić R, Ebling B, 

Ebling Z, Marković I, Šamija M. Efforts in fighting 

against cancer in Croatia have to be focused on the 

primary health care. Coll Antropol. 2008; 32:709- 

24. 

17. Ebling Z, Hadžić N. Uloga i prikladnost testiranja 

okultnog fekalnog krvarenja u programu zaštite od 

kolorektalnog carcinoma. Liječ Vjesn 1989; 111: 

432-6. 

18. Ebling Z.Hemoccult test sensitivity and specificity. 

Acta med Croat 2001; 55 (suppl 4):13-87. 

19. Ebling B, Ebling Z, Kovačić L, Vlahušić A, Tokalić 

M, Glavina K, Šerić V. Present state and possibilities 

for improvement of cancer prevention and early 

detection in the Osijek-Baranja County. Coll Antropol 

2005; 29:1-10. 

20. Strnad M, Ebling Z, Šamija M, Majnarić Lj, Gmajnić 

R, Ebling B, Pribić S. Launching of the National 

Program of the Prevention and Early Detection of 

colorectal cancer in Croatia and the Osijek-Baranja 

County. ESMO International Symposium:10th 

World Congress on Gastrointestinal Cancer, Barcelona, 

25-28 June 2008. Ann Oncol 2008; 19(suppl 

6):vi83, p.:216. 

21. Ebling Z, Strnad M, Šamija M, Marković I, Gmajnić 

R, Ebling B. Croatian National colorectal cancer early 

detection program. In:WONCA, editors. Abstract 

book of the WONCA Europe conference; Istambul, 

Turkey, September 13-15 2008. Istambul: WONCA, 

2008, pp.: 77-78. 

22. Ebling Z, Jakšić Ž, Santo T, Budak A, Eljuga D, 

Šerić V. New knowledge as a stimulus for action. 

In: UICC, editors. Proceedings of the XVI International 

Cancer Congress; New Delhi, India, October 

30 - November 5, 1994. New Delhi: Monduzzi 

Editore,1994: pp.: 2953-56. 

23. Ebling Z, Majnarić LJ, Gmajnić R, Ebling B. Towards 

cancer prevention in Croatia - Program of the City 

of Osijek League Against Cancer. In: UICC, eds. 

Proceedings of the UICC World Cancer Congress; 

2006 July 8-12, Washington, USA. Washington: Medimond, 

2006, pp.: 179-84. 

24. Katić M, Mazzi B, Petric D. Uloga liječnika obiteljske 

medicine u provedbi nacionalnog programa 

prevencije i ranog otkrivanja raka. In: Šamija M, 

Strnad M, Ebling Z,editors. Kako spriječiti i rano otkriti 

rak? Zagreb:Hrvatsko onkološko društvo HLZ, 

2007, pp.:77-82. 

25. WHO Programme on Cancer Control 2003 http:// 

www.who.int/cancer/main (April12 2007) 

26. The World Cancer Declaration 2006. International 

Union Against Cancer www.uicc.org/ 

27. Cancer Prevention and Control Resolution at the 

58th WHA on May 2005. Global WHO Cancer 

Control Strategy. http://www.who.int/mediacentre/ 

factsheets/fS297 (April 2007) 

28. American Cancer Society. Prevention and early detection. 

American Cancer Society Guidelines for the 

Early Detection of Cancer. www.cancer.org/docroot/ 

PED/content/PED_2_3X_ACS_Cancer_Detection_ 

Gu (Feb 8 2008). 

29. The Council of the European Union. Council Recommendation 

of 2 December 2003 on cancer screening 

(2003/878/EC) http://eur- 

30. lex.europa.eu/smartapi/cgi/sga_doc?smartapi!celex 

api!prod!CELEXnumdoc&lg=en&numdoc=32003 

H0878&model=guichett. (June 2008) 

31. IUCC/international union against cancer: roadmap for 

a European guideline in the fight against colon cancer. 

http://www.iucc.org/index.php?option=com_co 

ntent&task=view&id=15745&Itemid= (Oct 2007) 

32. Moss S. Evaluation and monitoring of screening 

programmes. In: Sankila R, Demaret E, Hakama M, 

Lynge E, Schoutan Lj, Parkin DM, eds. Evaluation 

and monitoring of screening programmes. European 

Commission. Europe against cancer program. Brussels-Luxemburg: 

European Commission, 2000. 

33. Wender RC. Preserving Primary Care: the Front 

Line in the War Against Cancer. CA Cancer J Clin 

2007; 57:4-5. 

34. Sarfaty M, Wender R. How to Increase Colorectal 

Cancer Rates in Practice. CA Cancer J Clin 2007; 

57:354-66. 

35. Jepson R, Weller D, Alexander F, Walker J. Impact 

of UK colorectal screening pilot on primary care. Br 

J Gen Pract 2005; 55:20-5. 

36. Segnan N, Senore C, Andreoni B, Arrigo A, Bisanti 

L, Cardelli A, Castiglione G, Crosta C, DiPlacido R, 

Ferrari A, Ferraris R, Ferrero F, Fracchia M, Gasperoni 

S, Malfitana G, Recchia S, Risio M, Rizzetto M, 

Saracco G, Spandre M, Turco D, Turco P, Zappa M; 

SCORE2 Working Group-Italy. Randomized trial of 

different screening strategies for colorectal cancer: 

patient response and detection rates. J Natl Cancer 

Inst 2005; 97:347-57.


Physicians overestimate patient’s knowledge of the process of 

informed consent: a cross-sectional study 

Marko Jukic 1 , Slavica Kozina 2 , Goran Kardum 3 , Rosemary Hogg 4 , Slavica Kvolik 5,6 

1 2 Department of Anaesthesiology and Intensive Care, Split University Hospital Centre, Split, Department of Psychological Medicine, Split 

University School of Medicine, Split, 3Department of Psychology, Split University Faculty of Humanities, Split, Croatia; 4Department of 

Anaesthetics and Intensive Care Medicine, Queens University Belfast, UK, 5Department of Anaesthesiology and Intensive Care, Osijek 

University Hospital Centre, 6School of Medicine, J.J. Strossmayer University of Osijek; Osijek, Croatia 


Slavica Kvolik 

Department of Anaesthesiology and 

Intensive Care, 

Osijek University Hospital Centre, 

School of Medicine, 

J.J. Strossmayer University of Osijek; 

Osijek, Croatia 

Phone: +385 31 511 502; 

Fax: +385 31 512 222; 

Email: s.kvolik@mefos.hr 


09 June 2010; 


14 August 2010; 

Accepted: 

31 August2010 

ABSTRACT 


Aim To evaluate the differences in the knowledge and attitudes of 

physicians and patients regarding the informed consent process. 

Methods After institutional approval was obtained cohorts of 269 

physicians and 265 patients completed a voluntary multiple-choice 

questionnaire on the informed consent process. 

Results Most of the responses between physicians and patients 

were significantly different. A total of 77 physicians (30.7%) reported 

that they personally informed patients about their medical 

condition and forthcoming clinical procedures in detail and 138 

(55%) informed patients as much as necessary. Only 29 patients 

(11%) reported being informed in detail, and 186 (70.2%) reported 

that they received only basic information (P

40 


INTRODUCTION 

The informed consent process involves communication 

between a physician and a patient. It 

should not be a passive and unilateral procedure 

in which a medical decision is left to the discretion 

of the physician, but an interactive process 

whereby patient’s preferences regarding medical 

decisions are considered (1-5.). Without official 

patient consent and essential communication 

with their physician, the principles of informed 

consent and patients’ rights would not be honoured 

with regard to diagnostic and treatment procedures, 

potential risks and complications and possible 

treatment alternatives concerning medical 

procedures (2). The completion of a consent form 

is only one part of the informed consent process, 

which also consists of discussions between patients 

and physicians regarding any proposed medical 

procedures (3,4). A patient’s signature on an 

informed consent form is necessary to initiate the 

treatment procedures, however, signing the consent 

form does not confirm a patient’s complete 

and correct understanding of the issues surrounding 

a medical procedure (1). 

Over the past three to four decades, medical ethicists 

have argued that patients should have a role 

in medical decision making (1). Due to efforts by 

professional medical organisations and lawmakers, 

patients now more consistently receive information 

concerning diagnostic and therapeutic 

procedures, risks, complications, and alternative 

methods of treatment (2, 4, 5). Even though the 

informed consent process has now been in use 

for a number of years, many patients still do not 

receive complete or desired information (2). 

In numerous studies, physicians have expressed a 

consistently positive attitude toward patient participation 

in the decision-making process (3,4). 

Despite this, however, patients’ understanding of 

their plan of care often remains limited (5). This 

insufficient knowledge may impair their ability 

to make important decisions regarding their hospital 

treatment (5). 

The aim of this study was to determine the differences 

in knowledge between physicians and patients 

regarding the informed consent process for 

invasive procedures and to compare their personal 

perception on the range of information given and 

obtained during the informed consent process. 

METHODS 

Study sample 

After obtaining institutional review board approval 

from the Ethics Committee in the Split University 

Hospital Centre from April to June 2006 

the survey was conducted among physicians performing 

invasive procedures at four hospitals in 

south Croatia, including Split University Hospital 

Centre, Zadar General Hospital, Dubrovnik 

General Hospital, and Sibenik General Hospital. 

Patients who were scheduled for invasive procedures 

in general anaesthesia were interviewed at 

the same time. Prior to study inclusion, all participants 

were informed about the purpose of the 

study and that participation was voluntary and 

anonymous. 

Data collection 

A self-administered questionnaire containing 

33 multiple-choice questions was given to 475 

physicians performing invasive procedures in 

general anesthesia in the departments of anaesthesiology, 

surgery, gynaecology, urology, orthopaedics, 

ophthalmology, otorhinolaryngology 

and internal medicine at the four hospitals. The 

physicians were asked to complete the questionnaire 

and return it within two months to the anaesthesiologist 

in charge of collecting completed 

questionnaires at each hospital. 

Three hundred consecutive elective adult patients 

undergoing invasive procedures at the Split University 

Hospital Centre during the study period 

were also asked to participate in the study during 

preanesthetic visit before invasive procedures. Of 

the 300 eligible patients, 265 (88.3%) agreed to 

a structured interview with an anaesthesiologist 

who read the questions aloud and recorded their 

answers. There were 145 patients interviewed at 

the Department of Surgery, 43 at the Department 

of Ophthalmology, 32 at the Department of Gynaecology, 

18 at the Department of Urology, 19 at 

the Department of Orthopaedics, and eight patients 

at the Department of Otorhinolaryngology. 

Questionnaires 

We developed physician and patient multiplechoice 

questionnaires containing questions related 

to the informed consent process, i.e. provision 

of information to patients, respecting patient

autonomy, knowledge of regulations and assessment 

of patient competence. The questionnaires 

were previously pilot-tested among 50 subjects 

and revised to create the final versions. After the 

pilot testing the physicians were given a questionnaire 

consisting of 33 multiple-choice questions 

(6). Finally, 18 questions appropriate for both 

physicians and patients were extracted, tested, 

and thereafter compared. 

Most of the questions on the physician and patient 

questionnaires were similar; however, the 

questions were rephrased to ask about experiences 

specific to either physicians or patients. For 

example, we asked physicians: “Where did your 

patient sign the treatment consent form?” This 

question was rephrased for patients as: “Where 

did you sign the treatment consent form?” 

Patients were given instructions to refer to the informed 

consent for procedure they are currently 

prepared for, whereas physicians were asked to refer 

to their last obtained informed consent process. 


Data were presented as frequencies and percentages 

in a tabulated format. Differences between 

categorical variables in each group were identified 

with the chi-square test and considered significant 

at P

42 


Patients’ knowledge about medical condition and 

medical procedures 

Physician and patient perceptions regarding patients’ 

knowledge about their medical condition 

and forthcoming medical procedures are illustrated 

in Table 2. 

Table 2. Patients’ knowledge concerning their medical condition 

and forthcoming treatment procedures 

Multiple-choice question/ 

statement 

No. of respondents (%) 

Physicians 

(n=251) 

Patients 

(n= 265) 

1. I inform patients about their medical condition and treatment 

procedures / I was informed about my condition 

in detail 77 (30.7) 29 (10.9) 

as much as necessary 138 (55.0) 186 (70.2)

patients and physicians to all the questions comparing 

their experiences regarding the procedure 

of obtaining informed consent to treatment (p< 

0.001). Disagreement was the most prominent 

in the question on the amount of the information 

presented to the patients before they had to make 

their decision on the forthcoming procedures. In 

the instance that patients were not able to make 

their own decision regarding treatment, most of 

them would leave the decision to physicians. On 

the contrary, physicians reported that, in such cases, 

they would routinely ask for consent from 

the patient’s family. 

DISCUSSION 

Recently Dieterich have identified a number of 

issues including law, ethics, knowledge, information, 

structural health care problems and funding 

issues as major areas of importance within 

a particular physician-patient interaction (7). 

The results of our study have shown significant 

differences in the knowledge and perception of 

these points of interests defining patient-doctor 

interaction between two study groups. Physicians 

lacked awareness about their professional, legal, 

and ethical obligations to provide patients with 

information concerning their medical condition 

and forthcoming diagnostic and therapeutic procedures. 

Accordingly, most patients reported receiving 

only limited or incomplete information, 

or in some cases no information obtained at all, in 

both this and numerous other studies (8). 

There was a limited amount of information that 

could be shared during the physician-patient consultation 

and this may occur because physicians 

often feel pressed for time. The majority of physicians 

in our study believed that patients received 

sufficient information to be able to make an informed 

decision on the recommended treatment. 

Patients expected to receive more detailed information, 

even though they were adequately informed 

about the risks, complications, and alternative 

methods of treatment (8,9). Other studies have 

shown that, after either two weeks or six months, 

patients do not seem to remember some information 

on the risks or alternative treatment methods 

(9). In addition, patients often do not wish to be 

fully informed of the risks and possible complications 

of the forthcoming surgery (10). 

Although many patients felt that they were gi- 

Jukić et al Physicians overestimate patient’s knowledge 

ven an insufficient amount of information, they 

still appreciate the input from medical staff. In 

situations where patients were unable to chose a 

treatment method and participate in the informed 

consent process, they were willing to trust their 

doctors with medical decisions. The most patients 

in our study reported providing their consent 

independently and agreeing on the treatment method 

suggested by their physicians. Levinson and 

co-workers in their population-based survey have 

obtained similar results to those observed in this 

study (11). They confirmed that nearly all respondents 

(96%) preferred to be offered choices and 

their opinions considered, but half of the respondents 

(52%) preferred to leave final decisions to 

their physicians. Furthermore, 44% of patients 

have preferred to rely on physicians for medical 

knowledge, and did not want to participate in the 

decision making process (11). In the era of widespread 

internet access, a significant amount of 

health information is now available to the general 

public, but the inability of patients to understand 

which information is useful have made them 

more likely than ever to trust their doctors (12, 

13). In a recent survey, Hesse et al confirmed that 

patients performing their own internet research 

are more likely to want to talk with their doctors 

about the treatment methods and that the internet 

does not replace the role of doctors in patient’s 

lives (12). Written information, video-recordings 

or web-based information might decrease decisional 

conflict and facilitate decision making, but 

are not substitutes for patient - physician communication 

(12-14). This is because during interpersonal 

discussion, decisions may vary depending 

on the nature of the procedure and the relevant 

comorbidities of the patient (15). A plain language 

should be used according to the cognitive abilities 

and education of the patient (1). 

Our study has shown that patients in South Croatia 

want their doctors be more involved in the 

informed consent process. On the contrary more 

doctors are prone to delegate such procedures to 

other members of medical stuff such as nurses or 

administrative personnel. Such a practice should 

be avoided as it is contrary to the code of medical 

ethics (4,16). Prior to obtaining consent, physicians 

should initiate a discussion about the diagnosis 

and treatment procedures, provide information 

concerning the potential risks and complications, 

43

44 


and review possible alternative methods of treatment. 

A signed consent form must be obtained 

by the patient’s physician, otherwise the signature 

on the consent form is nearly symbolic, possessing 

little value and may be legally and ethically 

discarded. 

In our study, patients did not find discussions regarding 

treatment costs justified or important before 

the treatment in public hospitals. As new treatment 

modalities are now available, and because 

patients and their families will be more engaged 

in the continuous treatments that may prolong after 

their discharge from hospital, this issue must 

be discussed before treatment has started. This is 

particularly important for therapies that are not 

paid by health insurance systems (16). 

This survey has several limitations. Firstly, a question 

regarding the completed level of education 

of the patient was excluded from the questionnaire. 

We were therefore unable to assess how the 

educational level of the patient influenced their 

ability to understand the provided medical information 

during the consent process. Physicians 

believed that only half of their patients completely 

understood the information provided that 

was needed to decide on treatment. Under that 

circumstances patient’s signature on the infor- 

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med consent form is legally and ethically questionable 

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family members. 

Another limitation of this study is that we interviewed 

only surgical patients, whereas not all of the 

physicians who completed the self-administered 

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benefit from upgrading informed consent procedures. 

Accordingly, consent forms for different 

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patient participation. Procedure specific 

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to make informed and autonomous choices 

and require physicians to inform patients about 

all aspects of specific procedures, be they routine 

or invasive (18,19). 


The study was performed as part of a research 

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No. 0141020, and was funded by a grant from the 

Ministry of Science, Education and Sport of the 

Republic of Croatia. 


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patients want to participate in decision making: a national 

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NK, Rimer BK, Viswanath K. Trust and sources of 

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DM, Groenewegen PP. How do healthcare consumers 

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aspx?rid=42830 (07.07. 2010) 

15. Paterick TJ, Carson GV, Allen MC, Paterick TE. 

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45

46 


Antibakterijski učinak materijala za punjenje korijenskih kanala, 

podloge i ispune kaviteta 

Suzana Ferk 1 , Paris Simeon 2 , Goranka Prpić Mehičić 2 , Smilja Kalenić 3 , Ivica Anić 2 , Silvana Jukić 2 

1 2 Privatna ordinacija dr. Ferk, Zagreb; Zavod za endodonciju i restaurativnu stomatologiju, Stomatološki fakultet Sveučilišta u Zagrebu, 

3Zavod za mikrobiologiju, Medicinski fakultet Sveučilišta u Zagrebu; Zagreb, Hrvatska 


Silvana Jukić, 

Zavod za endodonciju i restaurativnu 

stomatologiju, 

Stomatološki fakultet Sveučilišta u 

Zagrebu, 

Gundulićeva 5, 10000 Zagreb, Hrvatska 

Phone: +385 1 4802 126; 

fax: +385 1 4802 159 

E-mail: jukic@sfzg.hr 


27. srpanj 2009.; 


18. veljača 2010.; 

Prihvaćeno: 

12. travanj 2010. 

SAŽETAK 


Cilj Ispitati antibakterijski učinak materijala za endodontsku opskrbu 

zuba. 

Metode Testom difuzije u agaru ispitivana su punila korijenskih 

kanala Ketac Endo, AH Plus, Diaket i gutaperka. Od materijala za 

podloge ispitivani su Cink Oksid/Eugenol cement, staklenoionomerni 

cementi Fuji II LC Improved i Ketac Cem, te Phosphatcement 

i Harvard cement. Kao materijali za definitivne ispune ispitivani 

su amalgam, Ketac Molar i Fuji II LC Improved. Pri tome 

su rabljene bakterije Streptococcus mutans, Streptotoccus mitis, 

Lactobacillus species, Staphylococcus aureus, Enterococcus faecalis, 

te polimikrobna suspenzija. Za svaku ispitivanu bakteriju na 

inokulirane ploče krvnog agara raspoređeni su uzorci materijala. 

Rezultati Antibakterijski učinak imali su slijedeći materijali: punila 

kanala - Diaket, AH Plus, Ketac Endo, a za podloge: Cink Oksid/Eugenol 

cement, Phosphat i Harvard cement. Antibakterijski 

učinak nisu pokazali amalgam, gutaperka, Fuji II LC Improved, 

Ketac Cem i Ketac Molar. Diaket je pokazao statistički značajno 

jači antibakterijski učinak naspram AH Plusa, Ketac Endoa i gutaperke 

(p s.mitis

UVOD 

Jedan od glavnih ciljeva endodontskog liječenja 

jeste uklanjanje mikroorganizama iz korijenskog 

kanala što se nastoji postići instrumentacijom i 

irigacijom (1). Međutim, potpuno uklanjanje bakterija 

iz endodontskog sustava gotovo je nemoguće 

zbog složenog sustava lateralnih kanalića, 

anastomoza, bifurkacija i zavoja gdje su bakterije 

zaklonjene (2). Pored toga, dentinski kanalići i cement 

također su mjesta u kojima bakterije mogu 

perzistirati (1, 3). Zbog toga je važno da sredstvo 

za punjenje kanala ima antibakterijski učinak koji 

će pomoći u smanjenju broja mikroorganizama, te 

spriječiti neuspjeh endodontskog zahvata. Osim 

toga, poželjna su i antibakterijska svojstva materijala 

za podloge jer je dokazano da, ukoliko se 

zub nakon endodontskog zahvata odgovarajuće 

ne opskrbi, u vremenskom periodu od najviše tri 

mjeseca dolazi do propuštanja bakterija i gljiva 

(4, 5). Materijali za podloge i ispune trebali bi pored 

učinka mehaničke barijere i kemijskim putem 

djelovati na prodor mikroorganizama (1). 

Antibakterijski učinak materijala za opskrbu 

tvrdih zubnih tkiva može se odrediti testom difuzije 

u agaru, testom dilucije agara i direktnim 

kontaktnim testom. Test difuzije u agaru (agar 

diffusion test, ADT) najčešće je rabljena tehnika 

za procjenu antibakterijskih svojstava dentalnih 

materijala (6, 7). Provodi se postavljanjem određene 

količine ispitivanih materijala na ploču s 

krvnim agarom na kojem su nasađeni određeni 

sojevi bakterija. Rezultati se očitavaju mjerenjem 

i usporedbom zona inhibicije rasta bakterija izraženih 

u milimetrima (8). Manjkavost testa je da 

nejednaka fizikalna svojstva materijala utječu na 

njihovu difuziju kroz agar, pa rezultati istraživanja 

ne moraju odgovarati stvarnom antibakterijskom 

učinku (9). 

U ovom radu smo istražili antibakterijsko djelovanje 

materijala za endodontsku opskrbu zuba 

(punjenje kanala, podlogu i ispun) uporabom 

polimikrobnog markera načinjenog kombinacijom 

pet različitih sojeva fakultativnih anaerobnih 

bakterija i na svaki soj zasebno testom difuzije 

agarom. 

MATERIJAL I METODE 

U radu je ispitivan antibakterijski učinak slijedećih 

materijala za punjenje korijenskih kanala: 

Ferk et al Antibakterijski učinak endodontskih materijala 

AH Plus (Dentsply, De Trey, D-78467 Konstanz, 

Njemačka), Diaket (3M/ESPE, Seefeld, Njemačka), 

gutaperka štapića (SybronEndo, Amersfoort, 

Nizozemska), Ketac Endo (3M/ESPE, Seefeld, 

Njemačka). Od materijala za podloge ispitivani 

su: Ketac Cem (3M/ESPE, Seefeld, Njemačka), 

Phosphat Cement (Bayer Dental, Leverkusen, 

Njemačka), Fuji II LC Improved (GC, Tokyo, 

Japan), Harvard cement (Richter and Hoffmann 

Harvard Dental, Berlin, Njemačka) i Cink Oksid/ 

Eugenol cement (Speiko, Munster, Njemačka); 

a od materijala za ispune: Fuji II LC Improved 

(GC, Tokyo, Japan), Ketac Molar (ESPE, Seefeld, 

Njemačka) i amalgam bez cinka (Amalcap, 

Vivadent, Schaan, Lihtenštajn). 

Antibakterijski učinak ispitivan je pomoću pet 

sojeva fakultativno anaerobnih bakterija, pojedinačno 

i pomiješanih tako da tvore polimikrobnu 

suspenziju koja simulira uvjete in vivo (4), te su 

testirani uzorci materijala i na svaki bakterijski 

soj zasebno. 

Bakterije rabljene u ispitivanju bile su Streptococcus 

mutans 6715 WT, Streptotoccus mitis, 

Lactobacillus species NCTC1723, Staphylococcus 

aureus ATCC 29213 i Enterococcus faecalis 

ATCC 29212. 

Polipropilenske Ependorf tubice, volumena 1,5 

ml (Sigma, Aldrich, SAD), prerezane su i od njih 

su učinjeni standardizirani prstenovi promjera 3 

mm koji su poslužili kao kalupi za ispitivane materijale. 

Prstenovi su autoklavirani u autoklavu (Vacuclav 

24B+, Melag, Berlin, Njemačka) na temperaturi 

od 120°C i tlaku od 300 kP, tijekom 30 minuta. 

Ispitivani materijali zamiješani su prema naputku 

proizvođača i stavljeni u prstenove. 

Sterilni prstenovi, uronjeni u tekućinu eugenola 

ZnOE cementa, služili su kao pozitivna kontrola, 

dok su sterilni prestenovi postavljeni na ploče 

krvnog agara, inokulirani kontaminiranim bujonom, 

služili kao negativna kontrola. 

Ispitivane bakterije, prethodno kultivirane na pločama 

krvnog agara, inokulirane su u Shedlerov bujon 

i ostavljene preko noći u termostatu na 37 ºC. 

Na dan eksperimenta učinjeno je razrjeđenje 

bakterija (9 x 108 CFU/mL) u svježem mediju 

(McFarland 3 - McFarland Standard, Barium sulfate 

suspensium, bio Merieux sa, 69280 Marcy 

l’Etoile, France). 

47

48 


Za test difuzije u agaru 1/10 mL svake bakterijske 

kulture nasađeno je na ploče krvnog agara i 

ravnomjerno raspoređeno preko cijele površine 

ploče sa sterilnom ezom. Isto je učinjeno i s polimikrobnim 

markerom. 

Pripremljeni uzorci ispitivanih materijala iz kalupa 

postavljeni su na površine ploča krvnog 

agara s razmakom od najmanje 2 cm jedan od 

drugog. Ploče su inkubirane tijekom 48 sati na 

37 ºC u bubnju s anaerobnim uvjetima s Gas-Pakom 

(BBL, Anaerobic System Envelopes, Becton 

Dickinson and Company, Cockeysvill, MD 

21030 USA). 

Testirano je 12 replika za svaki ispitivani materijal. 

Antibakterijski učinak određen je mjerenjem 

zone inhibicije u mm. 

Rezultati antibakterijskog učinka, izraženi u milimetrima 

promjera zone inhibicije rasta bakterija, 

statistički su obrađeni, a postojanje razlika testirano 

je One and Two-Way analizom varijance 

(Anova) i Schefféovim post hoc testom. 

REZULTATI 

Antibakterijski učinak materijala za punjenje 

korijenskih kanala 

Srednje vrijednosti zone inhibicije rasta bakterija 

i standardne devijacije za ispitivane bakterije prikazane 

su u tablici 1. 

Analizom varijance uspoređen je antibakterijski 

učinak (tj. promjer zone inhibicije u milimetrima) 

materijala za punjenje korijenskih kanala 

Ketac Endo, AH Plus, Diaket i gutaperka. Antibakterijski 

učinak ovih materijala statistički 

se je značajno razlikovao kod svih šest ispitanih 

vrsta bakterija (p

N 

Streptococcus 

mitis 

Homogene 

podskupine* 

Streptococcus 

mutans 

Homogene 

podskupine* 

AH Plus i Ketac Endo, dok Gutaperka uopće nije 

pokazala antibakterijski učinak. 

Antibakterijski učinak materijala za podlogu 

Srednje vrijednosti i standardne devijacije zone 

inhibicije ispitivanih materijala za podlogu prikazane 

su u tablici 2. 

Analizom varijance uspoređen je antibakterijski 

učinak slijedećih materijala za podlogu: Harvard 

cement, Phosphat cement, Ketac Cem, Cink Oksid/Eugenol 

(ZnOE) i Fuji LC II Improved. 

Antibakterijski učinak navedenih materijala 

statistički značajno razlikovao se kod svih 5 

ispitanih vrsta bakterija i polimikrobne suspenzije 

(p

50 


Sva tri ispitivana cementa za punjenje korijenskog 

kanala, Ketac Endo, AH Plus i Diaket, 

imali su statistički značajan antibakterijski učinak 

na ispitivani soj Streptococcus mitis. Pri tome je 

Diaket pokazao najjači antibakterijski učinak. 

To se može objasniti poznatim antibakterijskim 

učinkom cinkovog oksida kojeg Diaket ima u 

svom sastavu (12). 

Isti rezultati dobiveni su i za ispitivane sojeve 

Streptococcus mutans, Lactobacillus achidophilus, 

Enterococcus faecalis i Staphylococcus aureus. 

Kod polimikrobne suspenzije materijali za punjenje 

kanala, s obzirom na svoj antibakterijski učinak, 

raspodijeljeni su na način kako su raspodijeljeni 

i kod bakterija pojedinačno. Ovi su rezultati 

u suglasnosti s rezultatima Barkhordara (13) koji 

je ispitivao učinkovitost deset različitih materijala 

za punjenje korijenskih kanala, te je u svom 

istraživanju pronašao da Diaket naspram ostalih 

materijala pokazuje najsnažniji inhibicijski efekt 

na Streptococcus mutans. Antibakterijsku superiornost 

Diaketa naspram ostalih materijala za 

brtvljenje korijenskih kanala potvrdili su svojim 

istraživanjem i Pumarola sa sur. (8) testom dilucije 

agara na 120 sojeva Staphylococcus aureus. 

Snažniji antibakterijski učinak Diaketa u odnosu 

na AH 26 i Epiphanya prema Enterococcus faecalis 

dokazali su Bodrumlu i Semiz (14). 

Perez i sur. (15) su pronašli najveću zonu inhibicije 

rasta bakterija oko Endomethasone punila, 

što se objašnjava njegovom parafolmaldehidnom 

komponentom koja se brzo i lako širi u ispitivanom 

mediju. Upravo zbog paraformaldehida i 

kortikosteroidne komponente, Endomethazone je 

punilo koje se ne preporuča (12). Nakon njega 

najveći promjer zone inhibicije imao je AH 26, 

pa Sealapex. Sealapex, kao i većina cemenata 

temeljena na kalcijevu hidroksidu, vremenom se 

N 

Streptococcus 

mitis 

homogene 

podskupine* 

Streptococcus 

mutans 

homogene 

podskupine* 

Tablica 5. Deskriptivni parametri antibakterijskog učinka 

materijala za ispun 

Tablica 4. Rezultati Scheffeovog testa za antibakterijski učinak materijala za podlogu kod bakterije Streptococcus mitis, Streptococcus 

mutans,Lactobacillus achidophilus, Enterococcus faecalis, Staphilococcus aureus i polimikrobni marker 

Lactobacillus 

achidophilus 

homogene 

podskupine* 

Enterococcus 

faecalis 

homogene 

podskupine* 

Staphylococcus 

aureus 

homogene 

podskupine* 

polimikrobni 

markeri 

homogene podskupine* 

1 2 1 2 1 2 1 2 1 2 1 2 3 

aritmetička sredina promjera zone inhibicije (mm) 

Fuji LC II Improved 12 0,0 0,0 0,0 0,0 0,0 0,0 

Ketac Cem 12 0,0 0,0 0,0 0,0 0,0 0,0 

Harward 12 7,3 7,3 7,2 7,4 6,8 6,6 6,6 

Phosphatcement 12 7,4 7,6 7,3 7,6 7,1 7,1 

ZnOE 12 7,7 8,1 7,3 7,7 7,2 7,5 

N, broj mjerenja; *homogene podskupine obuhvataju skupine materijala između kojih ne postoji statistički značajna razlika 

materijal M s.d. N 

Streptococcus mitis Fuji LC II Improved 0 0 12 

Amalgam 0 0 12 

Ketac Molar 0 0 12 

Streptococcus mutans Fuji LC II Improved 0 0 12 



Lactobacillus achidophilus Fuji LC II Improved 0 0 12 



Enterococcus faecalis Fuji LC II Improved 0 0 12 



Staphylococcus aureus Fuji LC II Improved 0 0 12 



polimikrobni marker Fuji LC II Improved 0 0 12 



*M, aritmetička sredina promjera zone inhibicije (mm); s.d., standardna 

devijacija; N, broj mjerenja 

razgrađuju u korijenskom kanalu za razliku od 

cemenata koji se temelje na umjetnim smolama 

(Diaket, AH Plus) koji su manje topljivi u slini 

i vodi (16). 

S obzirom da ne postoji materijal za punjenje korijenskog 

kanala koji u potpunosti sprječava koronarno 

mikropropuštanje (4), u ovo su istraživanje 

uključeni i materijali koji se rabe za podloge. Uz 

to su ispitivani materijali koji se rabe za konačnu 

restauraciju jer cjelokupna endodontska opskrba 

zuba pridonosi manjem propuštanju i konačnom 

uspjehu endodontskog liječenja (1). Od materijala 

koji se rabe za trajne ispune endodontski tretiranih 

zuba, u ovom radu ispitivani su Fuji II LC 

Improved, Ketac Molar i amalgam. Niti jedan od 

njih nije pokazao antibakterijski učinak. 

Fuji II LC Improved i Ketac Molar spadaju u skupinu 

staklenih ionomera. Smatra se da je oslobađanje 

iona fluora primarni razlog antibakterijskog 

djelovanja (17, 18), no ne i jedini. Dodatni uzrok 

može biti kiselina koja čini sastavni dio cementa, 

a rabi se za kondicioniranje dentina i omogućuje 

njegovu adheziju. Po nekim teorijama antibakte-

ijski učinak staklenih ionomera uzrokuje cink 

u njegovu sastavu (19). Poznato je da cink ima 

veći antibakterijski učinak od fluorida (19). Boeck 

i sur. (20) su pronašli, ispitivanjem inhibicije 

rasta bakterija u tekućim kulturama, da stakleni 

ionomeri koji se polimeriziraju svjetlom imaju 

manji antibakterijski učinak nego kemijskostvrdnjavajući 

stakleni ionomeri. 

U ovom istraživanju Fuji LC II Improved i Ketac 

Cem nisu uopće pokazali antibakterijski učinak 

niti kod jedne ispitivane bakterije, niti kod polimikrobne 

suspenzije, za razliku od istraživanja 

Vermeerscha i sur. (21) koji su pronašli antibakterijski 

učinak svih staklenih inomera prema Streptococcus 

mutans. U istraživanju Lewinsteina sa 

sur. (22) Ketac Cem nije pokazao antibakterijsku 

aktivnost direktnim kontakt testom, za razliku 

LITERATURA 

1. Ørstavik D, Pitt Ford TR. Essential Edontology. 7. 

izd. Oxford: Blackwell Science Ltd; 2005: 106-30. 

2. Ricucci D, Siqueira JF Jr. Fate of the tissue in lateral 

canals and apical ramifications in response to pathologic 

conditions and treatment procedures. J Endod 

2010; 36:1-15. 

3. Peters LB, Wesselink PR, Buijs JF, van Winkelhoff 

AJ. Viable bacterial in root dentinal tubules of teeth 

with apical periodontitis. J Endod 2001; 27:76-81. 

4. Miletić I, Prpić-Mehičić G,Maršan T, Tambić-Andrašević 

A, Plesko S, Karlović Z, Anić I. Bacterial 

and fungal microleakage of AH26 and AH Plus root 

canal sealers. Int End J 2002; 35:428-32. 

5. Ferk S, Malčić A, Jukić S, Anić I, Šegović S, Kalenić 

S. Coronal microleakage of two root end filling materials 

using a polymicrobial marker. J Endod 2008; 

34:201-3. 

6. Kaplan AE, Picca M, Gonzales MI, Macchi RL, Molganti 

SL. Antimicrobial effect of six endodontic sealers: 

an in vitro evaluation. Endod Dent Traumatol 

1999; 15:42-5. 

7. Gomes BPFA, Pedroso JA, Jacinto RC, Vianna ME, 

Ferraz CCR, Zaia AA, de Souza-Filho FJ. In vitro 

evaluation of the antimicrobial activity of five root 

canal sealers. Braz Dent J 2004; 15:30-5. 

8. Pumarola J, Berastegui E, Brau E, Canalda C, 

Jime´nez de Anta M T. Antimicrobial activity of seven 

root canal sealers: Results of agar diffusion and 

agar dilution tests. Oral Surg Oral Path Oral Med 

1992; 74:216-20. 

9. Çobankara FK, Altinöz HC, Erganiş Kürşat K, Belli 

S. In vitro antibacterial activities of root-canal sealers 

by using two different methods. J Endodont 

2004; 30:57-60. 

10. Tanomaru-Filho M, Poliseli-Neto A, Leonardo MR, 

Silva LA, Tanomaru JM, Ito IY. Methods of experimental 

induction of periapical inflammation. Microbiological 

and radiographic evaluation. Int End J 

2005; 38:477-82. 

Ferk et al Antibakterijski učinak endodontskih materijala 

od Harvard cementa i Duralona, što je u skladu s 

ovim istraživanjem u kojem su Phosphat cement, 

Harvard cement i Cink Oksid/Eugenol pokazali 

antibakterijski učinak (p

52 


Antibacterial effect of the materials for the root canal filling, bases and material for 

the cavity fillings 

Suzana Ferk 1 , Paris Simeon 2 , Goranka Prpić Mehičić 2 , Smilja Kalenić 3 , Ivica Anić 2 , Silvana Jukić 2 

1 2 Dr. Ferk Private Dental Practice , Zagreb, Department of Endodontics and Restorative Dentistry, School of Dental Medicine, University 

of Zagreb, 3Department of Microbiology, School of Medicine, University of Zagreb; Zagreb, Croatia 

ABSTRACT 

Aim To assess antimicrobial effects of the materials used in the endodontic treatment of the teeth. 

Methods The following root-canal fillings were examined: Ketac Endo, AH Plus, Diaket, and guttapercha, 

by means of the agar diffusion test. As for the base materials, Zink Oxide / Eugenol cement, 

glass-ionomere cements Fuji II LC Improved, Ketac Cem, and phosphate cement and Harvard cement 

were investigated. Finally, of the materials for final cavity filling, amalgam, Ketac Molar and Fuji II 

LC Improved were tested. In the present research, the following bacteria were applied: Streptococcus 

mutans, Streptotoccus mitis, Lactobacillus species, Staphylococcus aureus, Enterococcus faecalis, and 

polymicrobial suspention. For each individual bacterium tested, material samples were placed on the 

inoculated plates of blood-agar. 

Results Antibacterial effects were confirmed in the following materials: root-canal fillings - Diaket, 

AH Plus, Ketac Endo; bases - Zink Oxide / Eugenol cement, phosphat cement and Harvard cement. 

No antibacterial effect was established in: amalgam, gutta-percha, Fuji II LC Improved, Ketac Cem 

and Ketac Molar. Diaket showed a statistically more significant antibacterial effect in comparison with 

AH Plus, Ketac Endo, and gutta-percha (p s.mitis

NOTES 

Utjecaj trajanja dijabetesa i neregulirane 

glikemije na nastanak retinopatije 

Salem Alajbegović1 , Azra Alajbegović2 , Jasmina 

Omerović1 , Adnan Mušanović1 , Elmedin Lačić1 1 2 Služba za unutrašnje bolesti, Kantonalna bolnica Zenica; Neuropsihijatrijska 

klinika, Klinički centar Univerziteta u Sarajevu; 

Bosna i Hercegovina 

Corresponding author: Alajbegović Salem, Služba za unutrašnje 

bolesti, Kantonalna bolnica Zenica, Crkvice 67, Zenica, Bosna i 

Hercegovina 

Phone: +387 32 405 133; fax: +387 32 405 534; 

E-mail: dr-alajbegovic@kbze.ba 

Originalna prijava: 03. april 2010.; Korigirana verzija: 09. juni 

2010.; Prihvaćeno: 31. august 2010. 


SAŽETAK 

Cilj ovog istraživanja bio je utvrditi utjecaj trajanja 

dijabetesa i glikemije na nastanak dijabetične 

retinopatije kod pacijenata s dijabetesom 

tipa 1 i 2, te učestalost retinopatije prema spolu. 

Pregledano je 278 dijabetičara 1999. i 2004. 

godine, a anketnim listom uzeti su određeni podaci 

i upisani rezultati glikemije natašte, HbA1c, glukozurije i ketonurije. Retinopatija je 1999. 

godine zabilježena kod 80 (28,78 %) bolesnika, 

a 2004. godine 187 (67,27) (p

54 


PACIJENTI I METODE 

U istraživanje su bili uključeni bolesnici oboljeli 

od šećerne bolesti tipa 1 ili tipa 2, životne dobi 

od 18 do 80 godina, oba spola, a koji su randomizirano 

odabrani iz skupine dijabetičnih bolesnika 

liječenih na Internom odjelu Kantonalne bolnice 

u Zenici i domovima zdravlja Zeničko-dobojskog 

kantona, u periodu 1999–2004. godine. 

Svi bolesnici uključeni u istraživanje prethodno su 

potpisali pristanak za učestvovanje u ispitivanju. 

Ispitivanjem je obuhvaćena skupina od 278 bolesnika 

kojima je ranije klinički i laboratorijski 

jasno dijagnosticiran dijabetes, i to 58 bolesnika 

s dijabetesom tipa 1, a 220 s dijabetesom tipa 2. 

Pregledi, anketiranje i predviđeno laboratorijsko 

testiranje izvršeni su u svih ispitanika 1999. i 

ponovno 2004. godine. Svi su ispitanici pod jednakim 

uvjetima pregledani, anketirani i svima su 

uzeti određeni laboratorijski nalazi, a dobijeni podaci, 

tokom obje godine ispitivanja, evidentirani 

su na istovrsnom anketnom listu pojedinačno za 

svakog bolesnika. 

Prema anketnom listu od bolesnika su uzeti demografski 

podaci (dob, spol, obrazovanost, zaposlenost), 

trajanje dijabetesa, liječenje dijabetesa 

(dijeta, inzulin, peroralni lijekovi, kombinirana 

terapija), izvršena su mjerenja tjelesne težine, svi 

bolesnici su klinički (anamneza, fizikalno) pregledani, 

te su uzeti uzorci krvi i urina za utvrđivanje 

glikemije natašte, glukoziliranog hemoglobina, 

glukozurije i ketonurije. Svim ispitanicima 

urađen je i pregled očnog dna. 

Dobijeni rezultati obrađeni su pomoću t-testa 

proporcije za zavisne promjenljive varijable 

(p

Studije iz Evrope i SAD-a ubjedljivo su potvrdile 

povezanost između prevalencije retinopatije i 

trajanja dijabetesa tipa 1, posebno nakon 15-20 

godina trajanja dijabetesa. U Wisconsin Epidemiologic 

Study of Diabetic Retinopathy (WESDR) 

nađeno je da u grupi s početkom dijabetesa tipa 1 

u mlađoj životnoj dobi prevalencija bilo koje vrste 

retinopatije progresivno je rasla od 2% u osoba s 

manje od dvije godine trajanja dijabetesa do 98% 

u onih s više od 15 godina trajanja bolesti. U grupama 

s kasnijim početkom bolesti prevalencija 

dijabetične retinopatije također je bila pozitivno 

povezana s trajanjem dijabetesa tipa 1 (4, 5). 

Studije američke populacije (4) našle su retinopatiju 

u skoro 100% pacijenata nakon više od 20 

godina trajanja dijabetesa, dok evropske populacije 

ipak izgleda imaju nešto nižu maksimalnu 

prevalenciju od 80-90% (13, 14). EURODIAB 

prospektivna studija komplikacija u dijabetesu 

tipa 1 danas je možda pribavila najbolje dokaze za 

faktore rizika koji doprinose razvoju retinopatije 

u ovoj bolesti. U sedmogodišnjem praćenju retinopatija 

je nađena u 56% slučajeva (7). Rezultati 

ovoga istraživanja u pogledu povezanosti i značaja 

trajanja dijabetesa za porast broja oboljelih od 

retinopatije saglasni su s pomenutim studijama. 

U pogledu regulacije glikemije, kao faktora 

nastanka dijabetične retinopatije, bolesnici su 

na početku ispitivanja bili loše regulirani jer je 

prosječna glikemija natašte tada iznosila 13,69 

mmol/L, a HbA 13,02%. Na kraju ispitivanja 

1c 

bolesnici su bili bolje regulirani jer je prosječna 

glikemija natašte u ispitivanom uzorku iznosila 

13,0 mmol/L, a HbA 10,57%. Međutim, to 

1c 

je još uvijek daleko od dobre regulacije. Usljed 

toga, ova neregulirana glikemija najvjerovatnije 

je faktor koji je utjecao na porast pojave dijabetične 

retinopatije u našem uzorku. 

U Wisconsin Eye Study pokazano je da su incidencija 

i progresija retinopatije bile u korelaciji s glike- 

Grafikon 1. Broj bolesnika s dijabetičnom retinopatijom 

mijskim statusom u bolesnika s dijabetesom tipa 1 i 

2 (12). Henricson i sar. na grupi osoba s dijabetesom 

tipa 2 pokazali su da je progresija retinopatije, prije 

i poslije hirurške intervencije na mreni, bila u korelaciji 

s prosječnim HbA c, trajanjem dijabetesa, 

1 

inzulinskim liječenjem i prisutnošću retinopatije na 

početku bolesti (15). U jednom ispitivanju Oklahoma 

indijanaca incidencija retinopatije bila je 

72,3%, a ustanovljeni neovisni prediktori retinopatije 

bili su glikemija natašte na početku ispitivanja, 

krvni pritisak i trajanje dijabetesa (16). 

I na početku i na kraju našeg istraživanja dijabetičari 

su bili loše regulirani. Nađen je značajan, 

više od dvostrukog, porast broja dijabetičara zahvaćenih 

dijabetičnom retinopatijom pet godina 

nakon prvog ispitivanja. Na temelju ovih rezultata, 

kao i rezultata istraživanja drugih autora, 

nameće se zaključak da su uloga i značaj neregulirane 

glikemije, kao i trajanje dijabetesa, imali 

veliki značaj u velikom porastu broja pacijenata 

zahvaćenih dijabetičnom retinopatijom. 

Dijabetična retinopatija je česta komplikacija u 

dijabetesu tipa 1 i predstavlja jedan od glavnih 

uzroka sljepila u radno aktivnog stanovništva u 

zapadnim zemljama (3-5), kao i u Bosni i Hercegovini 

(17). Isto tako, porast dijabetične retinopatije 

ukazuje na značaj metoda prevencije u 

nastanku retinopatije i sljepoće kod bolesnika s 

dijabetesom među kojima, pored opisanih, važno 

mjesto zauzimaju i redovni pregledi oftalmologa 

i pravovremeno i egzaktno indicirana laser-terapija 

(18, 19). 



postoji. 

LITERATURA 

1. 

2. 

3. 

Notes 

World Health Organization Consultation. Definition. 

Diagnosis and Classifitation of Diabetes mellitus an 

its Complications, Part I: Diagnosis and Classification 

of Diabetes Mellitus. Report of a WHO Consultation. 

Geneva: World Health Organization, 1999. 

Sjølie AK. Ocular complications in insulin treated 

diabetes mellitus: an epidemiological study. Acta 

Opthalmol 1985; Suppl.172:1-77. 

Sjølie AK, Green A. Blindness in insulin-treated diabetic 

patiens with age at onset < 30 years. J Chron 

Dis 1987; 40:215-20. 

55

56 


4. Klein R, Klein BEK, Moss SE et al. The Wisconsin 

epidemiologic study of diabetic retinopathy. 2. Prevalence 

and risk of diabetic retinopathy when age 

at diagnosis is less than 30 years. Arch Ophthalmol 

1984; 102:520-6. 

5. Klein R, Klein BEK, Moss SE et al. The Wisconsin 

Epidemiologic Study of Diabetic Retinopathy.III. 

Prevalence and risk of diabetic retinopathy when age 

at diagnosis is 30 or more years. Arch Ophthalmol 

1984; 102:527-32. 

6. Tamara Lecaire, Mari Palta, Hongling Zhang, Catherine 

Allen, Ronald Klein, Donn D’Alessio. Lowerthan-expected 

prevalence and severity of retinopathy 

in an incident cohort followed during the first 4-14 

years of type 1 diabetes: the Wisconsin Diabetes Registry 

Study. Am J Epidemiol 2006; 164:143-50. 

7. Chaturvedi N, Sjoelie A, Porta M et al. Markers of 

insulin resistance are strong risk factors for retinopathy 

incidence in type 1 diabetes. Diabetes Care 

2001; 24:284-9. 

8. Klein R, Klein BEK. Vision disorders in diabetes. In: 

Hamman R, Harris MWH, eds. Diabetes in America. 

US Public Healt Service NIH Pub. No. 85-1468, Vol. 

13. Bethesda, MD: NIH, 1983:1-36. 

9. Eshøj O, Green A, Borch-Johnsen K, Feldt-Rasmussen 

B, Beck-Nielsen H. Increased prevalence of 

insulin-treated diabetes mellitus in Funen County, 

Denmark. J Intern Med 1994; 235:405-10. 

10. The Diabetes Control and Complications Trial Research 

Group. The effect of intensive treatment of 

diabetes on the development and progression of 

long-term complications in insulin-dependent diabetes 

mellitus. N Engl J Med 1993; 329:977-86. 

11. Shaya TF, Aljawadi M. Diabetic retinopathy. Clinical 

Ophthalmology 2007; 1:259 – 65. 

12. Klein R, Klein BEK, Moss SE. Relation of glycemic 

control to diabetic microvascular complications in 

diabetes mellitus. Ann Intern Med 1996; 124:90-6. 

13. Nielsen NV. Diabetic retinopathy I. The course of 

retinopathy in insulin-treated diabetics: a one year 

epidemiological cohort study of diabetes mellitus: 

the island of Falster, Denmark. Acta ophthalmol 

1984; 62:256-65. 

14. Jerneld B. Prevalence of diabetic retinopathy: A population 

study from the Swedish island of Gotland. 

Acta Ophthalmol 1988; 66(Suppl 188):1-32. 

15. Henricsson M, Heijl A, Janzon L. Diabetic retinopathy 

before and after cataract surgery. Br J Ophthalmol 

1996; 80:789-93. 

16. Lee ET, Lee VS, Kingsley RM, Lu M, Russel D, Asal 

NR, Wilkinson CP, Bradford RH. Diabetic retinopathy 

in Oklahoma Indians with NIDDM. Incidence 

and risk factors. Diabetes Care 1992; 15:1620-7. 

17. Stolić V. Sljepoća usljed šećerne bolesti na području 

BiH. Savjetovanje o socio-oftalmološkim aspektima 

sljepoće. Zbornik radova. Sarajevo 1984. 67-75. 

18. Alimanović Halilović E. Laser u oftalmologiji. Dijabetična 

retinopatija. Sarajevo: Institut za istraživanje 

i razvoj, Klinički Centar Univerziteta u Sarajevu, 

2006.:1-124. 

19. Misita V. Dijabetična retinopatija liječenje laserfotokoagulacijom. 

Beograd: Zavod za udžbenike i nastavna 

sredstva, 2000:1-190. 

Influence of lasting diabetes and nonadjustable 

glycosylated haemoglobin on 

occurrence of retinopathy 

Salem Alajbegović1 , Azra Alajbegović2 , Jasmina 

Omerović1 , Adnan Mušanović1 , Elmedin Lačić1 1Department of Internal Diseases, Cantonal Hospital Zenica, 

2Neuropsychiatry Clinic, Clinical Center of Sarajevo University; 


ABSTRACT 

The aim of this study was to investigate the effect 

of the duration of diabetes and glycemia on the 

development of diabetic retinopathy in diabetes 

types 1 and 2 and the prevalence of retinopathy 

by sex. It examined 278 diabetics in 1999 and 

2004, a questionnaire was used to collect data 

and results of fasting glucose, HbA1c, glycosuria 

and ketonurie were recorded. Retinopathy was 

noted in 80 (28,78%) and 187 (67,27%) patients 

during 1999 and 2004, respectively (p

multiplom sklerozom, korišten je specijalno dizajniran 

upitnik i historije bolesti pacijenata koji 

su pod dijagnozom multiple skleroze liječeni na 

Neurološkoj klinici Kliničkog centra Univerziteta 

u Sarajevu (KCU), u periodu od 01. 01. do 31. 

12. 2006. godine. Ukupno je zabilježeno 71 (48 

žena i 23 muškarca) oboljelih. Infekcija kao precipitirajući 

faktor zabilježena je kod 21 (29,5%), 

a psihički stres kod 12 (16,9%) oboljelih, dok je 

kod 43 oboljela (60,56%) zabilježen RR tip bolesti. 

Terapija interferonom provodila se kod 9 

(12,67%), a visokim dozama metilpredinisolona 

kod 47 (66,7%) pacijenata. Depresivni poremećaj 

bio je prisutan kod 23 (32,9%), a kognitivna disfunkcija 

kod 7 (9,86%) bolesnika. Rezultati ovoga 

istraživanja, u kojem su prvi put na području 

Bosne i Hercegovine prikazane epidemiološke 

karakteristike multiple skleroze, upućuju na potrebu 

uspostavljanja registra oboljelih i obavezu 

pridržavanja terapijskih smjernica. 

Ključne riječi: multipla skleroza, kognitivna 

disfunkcija, depresija. 

UVOD 

Multipla skleroza (MS) je hronično autoimuno 

oboljenje centralnog nervnog sistema karakterisano 

propadanjem mijelinskog omotača, gubitkom 

oligodendrocita i oštećenjem aksona u raznim 

dijelovima mozga i kičmene moždine (1). 

Patofiziološki, multipla skleroza je kompleksno, 

u početku upalno, a kasnije i neurogenerativno 

oboljenje, kod kojeg kombinacija genetskih faktora 

i faktora okoline dovodi do nastanka upalnog 

procesa i posljedičnog oštećenja mijelina (2-4). 

Postoje četiri tipa toka bolesti: relapsirajuće-remitirajuća 

forma, sekundarno progresivna forma, 

primarno progresivna i progresivna MS s fazama 

pogoršanja. Dijagnoza se postavlja na osnovu 

kliničkog toka, MRI galve i imunogamaglobulina 

gama u likvoru (5). 

Liječenje multiple skleroze podrazumijeva tretman 

akutnih egzacerbacija, zaustavljanje napredovanja 

bolesti, simptomatsko liječenje, tretman 

komplikacija i pojedinih sindroma, kao i fizikalnorehabilitacijski 

tretman (6). Tretman akutnih egzacerbacija 

multiple skleroze fokusiran je na davanje 

visokih doza kortikosteroida (6), a imunomodulatori 

zaustavljaju napredovanje bolesti (7). 

Cilj našeg istraživanja bio je odrediti spolne i dobne 

karakteristike, tip bolesti, prosječno trajanje 

hospitalizacije, precipitirajuće faktore nastanka ili 

uvoda u pogoršanje bolesti, psihičke smetnje, te 

liječenje imunomodulatorima. Multipla skleroza 

je bolest posebnih karakteristika vezanih za podneblje, 

klimu i faktore okoline. Budući da slično 

istraživanje nije rađeno u regionu, rezultati ovoga 

istraživanja pomoći će zdravstvenim radnicima i 

bolesnicima da upoznaju bolest, njene specifičnosti 

i optimalno zbrinjavanje ovih bolesnika. 

ISPITANICI I METODE 

Za ovo istraživanje korištene su historije bolesti 

pacijenata, koji su pod dijagnozom multiple skleroze 

liječeni na Klinici za neurologiju Kliničkog 

centra Univerziteta u Sarajevu, u periodu od 01. 

01. do 31. 12. 2006. godine. Korišten je modificirani 

anketni listić, koji se inače koristi na Neurološkoj 

klinici Kliničkog centra Univerziteta u 

Sarajevu, a koji se sastojao od četiri dijela: opći 

demografski podaci o pacijentu (dob, spol, zanimanje), 

klinički parametri (simptomi, neurološki 

sindrom, stepen onesposobljenja, psihičke smetnje 

s posebnim osvrtom na depresiju i kognitivnu 

disfunkciju), dijagnostičke procedure i terapija. 

Dijagnoza MS-a ustanovljena je u skladu s Poser 

i McDonald kriterijima (8, 9). 

Neurološki status i progresija bolesti evaluirani 

su pomoću skale stepena onesposobljenja specifičnog 

za multiplu sklerozu prema Kurtzkeu kao 

EDSS (engl. expanded disability status scoring) 

(10). 

Statistička obrada podataka izvršena je pomoću 

računarskog programa Excel (Microsoft Office 

Excel 2003) i SPSS računarskog programa za statističke 

analize (SPSS - Statistical Package for 

Social Sciences), verzija 12.0. 

REZULTATI 

Notes 

Obrađena je ukupno 71 historija bolesti, odnosno 

slučajevi 48 žena i 23 muškarca (61,97%; odnosno 

38,03%), i to prema slijedećim dobnim skupinama: 

u dobnoj grupi 0-19 zabilježen je jedan 

pacijent (1,4%); u dobnoj grupi 20-29 zabilježeno 

je šest pacijenata (8,45%); a u dobnim grupama 

30-39, 40-49 i 50 i više godina, zabilježeno je 

22 (30,99%), 31 (43,66%), odnosno 11 (15,49%) 

pacijenata. 

Najveći broj pacijenata boravio je u bolnici 8-14 

dana, 31 (43,66%); 19 (26,7%) pacijenata bo- 

57

58 


ravilo je 15-30 dana; 11 (15,5%) 0-7 dana; a 10 

(14,1%) pacijenata boravilo je u bolnici duže od 

30 dana. 

Dvanaest pacijenata (8,45%) bilo je hospitalizirano 

jedanput; 32 (22,53%) dva puta; 16 (11,27%) 

tri puta; 28 (16,9%) četiri; po osam (5,63%) pacijenata 

pet i šest puta; a 36 (25,35%) pacijenata 

boravilo je u bolnici više od šest puta. 

Najveći broj pacijenata imao je simptome slabosti, 

59 (32,42%); a najmanji broj imao je simptome 

glavobolje, 9 (4,95%); simptome umora, 

vida, vrtoglavice i smetnje govora imalo je 39 

(21,43%); 33 (18,13%); 30 (16,48%); odnosno 

12 (6,59%) pacijenata. 

Najveći broj pacijenata imao je piramidalne 

znakove bolesti, 63 (40,65%); a najmanje ih je 

imalo smetnje sfinktera, 19 (12,26%); dok je 38 

(24,52%) i 35 (22,58%) pacijenata imalo simptome 

od strane kranijalnih nerava, odnosno cerebralne 

znakove. 

Najučestaliji tip MS-a u ispitivanoj grupi pacijenata 

bio je RR MS (relapsirajuće-remitirajući) 

u 43 (60,56%) pacijenta, dok je u 14 (19,72%) 

pacijenata ustanovljen SP MS (sekundarno progresivni 

tip bolesti). U 8 (11,25%) pacijenata 

tip bolesti nije bio poznat, dok P MS (primarno 

progresivni oblik sa pogoršanjima) i PP MS (primarno 

progresivni) nisu ustanovljeni kod naših 

ispitanika. 

Osam pacijenata (11,27%) imalo je dvije remisije; 

5 (7,04%) tri; 6 (8,45%) četiri; 12 (16,90%) 

pet i više remisija; dok kod 40 (56,34%) pacijenata 

broj remisija nije bio poznat. 

Najviše oboljelih od MS-a liječeno je vitaminima 

C, 52 (73,23%); E, 36 (50,70%); ili B, 31 

(43,46%); te cobalom, 20 (28,16%); methyprednisolonom 

i enkortenom po 18 (25,35%), odnosno 

18 (25,35%); lioresalom, 13 (18,30%); interferonom 

1-b, 9 (12,67%); te mitoxantronom, 2 

(2,81%) pacijenta. 

DISKUSIJA 

Multipla skleroza kao hronično, progresivno, 

neurološko oboljenje utiče na sve aspekte života 

oboljelih (11, 12). Značajno utiče na fizičko 

i psihičko zdravlje, finansijski i društveni status, 

te profesionalno i porodično funkcioniranje (13, 

14). Ovo oboljenje onesposobljava bolesnike u 

relapsima i u toku bolesti. 

Psihičke smetnje (anksioznost, depresija, kognitivna 

oštećenja, emocionalna nestabilnost) mogu 

biti manifestacija osnovne bolesti, ali i posljedica 

adaptacije na bolest (11, 12, 15). Kliničari najveću 

pažnju posvećuju fizičkim manifestacijama 

bolesti, no, međutim, pacijentima značajne probleme 

prave i kognitivna disfunkcija i afektivna 

simptomatologija (11). 

Učestalost multiple skleroze je u porastu, a češće 

obolijevaju osobe ženskog spola. U oko 80% 

slučajeva bolest počinje između 20. i 40. godine, 

u oko 9,5% slučajeva kod osoba starijih od 50 

godina, te u 5% slučajeva kod osoba mlađih od 

20 godina (15). Prosječna životna dob ispitanika 

našeg istraživanja iznosi 41,22 ± 9,3 godina, uporedno 

rezultatima Amata s 44,89 +/- 11,27 godina 

(15). 

Odnos muškaraca i žena u našem uzorku iznosio 

je 2:1 i sukladan je epidemiološkim podacima u 

drugim istraživanjima (15, 16). Srednja dob oboljelih 

u istraživanju Solarija i sar. (italijanska populacija) 

iznosila je 42,6 ± 11,2 i u potpunosti je 

uporedna našim rezultatima (16). Prosječan broj 

dana hospitalizacije naših ispitanika od 8-14 dana 

uporedan je rezultatima Vickreya i sur., čiji su pacijenti, 

hospitalizirani u prethodnoj godini, pokazali 

niže skorove fizičkog i mentalnog zdravlja u 

odnosu na nehospitalizirane (p < 0,05), a razlozi 

hospitalizacija su bili relapsi (pogoršanja) bolesti 

(17). Razlog pogoršanja bolesti kod naših pacijenata 

bile su ponovljene hospitalizacije, budući 

da je 81,65% pacijenata imalo više hospitalizacija 

s prosječnim trajanjem hospitalizacije od 19,5 

dana. U studiji Solarija i sur. prosječna dužina 

trajanja bolesti bila je nešto veća u odnosu na naš 

uzorak (12,8 +/- 8,9 godina) (16), dok su studije 

Idimana i Amata pokazale da je dužina trajanja 

bolesti imala signifikantan, obrnuto proporcionalan 

odnos s fizičkim i mentalnim skorovima kvaliteta 

života (12, 15). 

Premda se dizajn naše studije razlikovao od dizajna 

drugih studija, s obzirom da metodološki 

nismo ispitivali kvalitet života (12, 15), ipak naše 

rezultate možemo upoređivati s rezultatima navedenih 

autora pošto je cilj našeg istraživanja bio 

prikazati epidemiološke karakteristike populacije 

bolesnika s multiplom sklerozom iz dokumentacije 

koju smo imali na raspolaganju. Pogoršanju 

ili nastanku bolesti kod naših pacijenata najčešće 

je prethodila urinarna infekcija, zatim stres, što

se može objasniti često prisutnim poremećajima 

mikcije i stresnim načinom života kod bolesnika 

(11). 

Multiplu sklerozu karakteriše veliki broj neuroloških 

simptoma i znakova, ovisno o lokalizaciji 

lezija CNS-a, a osnovni su diseminacija u vremenu 

i prostoru (19). Najčešći simptom kojeg 

su imali naši pacijenti bila je slabost, a najrjeđi 

glavobolja; kao najčešći znak imali su piramidalne 

znakove, a najrjeđe smetnje sfinktera, što je 

u skladu s ranije objavljivanim rezultatima (19, 

20). 

Kognitivna disfunkcija ustanovljena je kod 10 

(14,08%) naših pacijenata, što je manje nego u 

drugim istraživanjima (11), dok je depresija bila 

registrovana kod 32 (45,07%) pacijenta. Razlog 

je najvjerovatnije u dizajnu retrospektivnog ispitivanja. 

Kognitivni deficit može se javiti u ranim 

fazama bolesti, te može doći do njegovog pogoršavanja 

s napredovanjem bolesti (11, 17). Istraživanja 

su ukazala da je kognitivno oštećenje u 

korelaciji s atrofijom korteksa, vidljivoj magnetnom 

rezonancom, što je valjan parametar praćenja 

bolesti (18). 

U našoj studiji 4,95% pacijenata izjasnilo se o 

prisustvu osjećaja boli u posljednje četiri sedmice. 

Ispitivanjem prevalence, intenziteta, uticaja 

i biopsihosocijalne korelacije prisutnosti bola, 

koje su Ehdea i sur. proveli među 449 osoba oboljelih 

od multiple skleroze, zaključeno je kako je 

četvrtina ispitanika imala problem hronične boli, 

karakteriziran kao intenzivan bol, uz signifikantnu 

interferenciju sa svakodnevnim aktivnostima 

(19). Bol kao simptom, u našem istraživanju, 

naveden je signifikatno manje često u odnosu na 

rezultate Ehdea i saradnika. 

Treba napomenuti i negativan uticaj osjećaja bola 

i prisustva sfinkterijalnih smetnji na svakodnevne 

aktivnosti i kvalitet života pacijenata (19). 

Sfinkterijalne smetnje različitog stupnja izraženosti 

imalo je 12,26% pacijenata, a Hajrić i saradnici 

objavili su slične rezultate (20). 

U terapiji MS-a, u našem uzorku, najčešće su 

korišteni lijekovi iz grupe simptomatskih lijekova. 

Methyprednisolon kao standardna terapija 

relapsa primjenjivan je kod 25,4% pacijenata, a 

imunomodulatori kod 12,7% pacijenata. Inače, 

terapija imunomodulatorima dostupna je malom 

broju pacijenata s obzirom na ograničena finan- 

sijska sredstva koja se daju iz fondova za ovu 

bolest, kao i vrlo dugu listu čekanja za terapiju 

(21). 

Rezultati ovog istraživanja, u kojem su prvi put 

na području Bosne i Hercegovine prikazane epidemiološke 

karakteristike multiple skleroze, upućuju 

na potrebu uspostavljanja registra oboljelih i 

obavezu pridržavanja terapijskih smjernica. 



postoji. 

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C, Rossi L. Quality of life in multiple sclerosis: the 

impact of depression, fatigue and disability. Mult Scler 

2001; 7:340-4. 

16. A. Solari, G. Filippini, L. Mendozzi, A. Ghezzi, S. 

Cifani, E. Barbieri, S. Baldini, A. Salmaggi, L. L. 

Mantia, M. Farinotti, D. Caputo, and P. Mosconi Validation 

of Italian multiple sclerosis quality of life 54 

questionnaire J Neurol Neurosurg Psychiatry 1999; 

67:158-62. 

17. Vickrey BG, Hays RD, Harooni R, Myers LW, Ellison 

GW. A health-related quality of life measure for 

multiple sclerosis. Qual Life Res 1995.4:187-206. 

18. Idiman E, Uzunel F, Ozakbas S, Yozbatiran N, Oguz 

M, Callioglu B, Gokce N, Bahar Z Cross-cultural 

adaptation and validation of multiple sclerosis quality 

of life questionnaire (MSQOL-54) in a Turkish 

multiple sclerosis sample J Neurol Sci 2006; 240:77- 

80. Ehde DM, Gibbonsd LE, Chwastiak L, Charles 

HB, Sullivan MD, Kraft GH. Chronic pain in a large 

community sample of persons with multiple sclerosis. 

Mult Scler 2003; 9:605-11. 

19. Hajric S, Alajbegovic A, Subasic N, Imamovic Dz, 

Nakicevic A. Quality of life in multiple sclerosis: relation 

to the clinical parameters of the disease. Med 

Arh 2008; 62:142-5. 

20. Alajbegović A, Denišlić M. Multipla skleroza. Treće 

prošireno i dopunjeno izdanje Sarajevo: Magistrat, 

2010: 55-87. 

Epidemiology of multiple sclerosis in 


AzraAlajbegović¹, Salem Alajbegović², Jasminka 

\elilović-Vranić¹ , 

Clinic of Neurology, Clinical Center of Sarajevo University¹, 

Cantonal Hospital Zenica² 

ABSTRACT 

In order to examine precipitating factors for 

occurrence of multiple sclerosis or inception of a 

relapse in patients suffering from multiple sclerosis 

a specially designed questionnaire was used, 

including history records of patients with multiple 

sclerosis treated at the Clinic of Neurology 

of the Clinical Center of Sarajevo in the period 

between January 1 st and December 31 st 2006. The 

number of patients with MS was 71 (48 women 

and 23 men). An infection as a precipitating factor 

was noted in 21 (29.57%) cases, stress was 

noted in 12 patients (16.9%) whereas 43 patients 

(60,12%) had the RR type of the disease. Nine 

patients were treated with interferon therapy 

(12.67%) and 47 patients (66.1%) with high doses 

of metilpredinisolone . Depression disorder 

was noted in 23 (32.9%) patients whereas 7 patients 

had cognitive dysfunction (9.86%). Results 

of this study, which have shown epidemiological 

characteristics of multiple sclerosis for the first 

time in Bosnia and Herzegovina, indicate that 

there is a need to create a unified register of patients 

and to request compliance with therapeutic 

guidelines. 

Key words: multiple sclerosis, cognitive dysfunction, 

depression 

Original submission: 05 June 2010; Revised submission: 21 

September 2010; Accepted: 26 October 2010 

NOTES 

Anxiety, splint treatment and clinical 

characteristics of patients with osteoarthritis 

of temporomandibular joint and 

dental students – a pilot study 

Tomislav Badel1 , Sandra Kocijan Lovko2 , Dijana 

Podoreški3 , Ivana Savić Pavčin4 , Josipa Kern5 1Department of Prosthodontics, School of Dental Medicine, 

University of Zagreb, Zagreb, 2Psychiatry Outpatient Department, 

General Hospital “Zabok”, Zabok, 3Department of Diagnostic and 

Interventional Radiology, Clinical Hospital “Sestre milosrdnice”, 

University of Zagreb, Zagreb, 4Department of Dental Anthropology, 

School of Dental Medicine, University of Zagreb, Zagreb, 

5Department of Medical Statistics, Epidemiology and Medical 

Informatics, School of Public Health “Andrija Štampar”, School 

of Medicine, University of Zagreb, Zagreb; Croatia 

Corresponding author: Tomislav Badel, Department of Prosthodontics, 

School of Dental Medicine, University of Zagreb, 

Gundulićeva 5, 10000 Zagreb, Croatia; Phone: +385 1 48 02 

125, Fax: +385 1 48 02 159; E-mail: badel@sfzg.hr 


ABSTRACT 

The aim of this study was to evaluate the use of 

splint treatment for therapy of osteoarthritis of 

temporomandibular joint, and to compare the 

level of anxiety (State-Trait Anxiety Inventory, 

STAI) and clinical characteristics between 16 

patients and 20 asymptomatic dental school students. 

Magnetic resonance imaging (MRI) was 

used for all subjects. Dental students showed a

statistically significant higher capacity of mouth 

opening (p

62 


the patients had reduced pain intensity (18.75%), 

and 2 (12.50%) of all the patients’ sample were 

without improvement of the TMJs condition. 

The median values of STAI scores for patients 

were also divided in two groups: patients with 

painless TMJs after splint treatment check-up and 

the patients who still had painful TMJs (Table 1). 

However, there was a statistically significant difference 

for STAI 1 scores only (Kruskal-Wallis 

test KW (df=1) =5.1822, with p=0.023). There 

was no difference in STAI 2 (Kruskal-Walis test 

KW (df=1) =0.3988, with p=0.528). Patients 

who had no improvement (still suffer pain) after 

splint treatment in the osteoarthritic TMJs had a 

higher value of anxiety by STAI tests, for STAI 

1 Kruskal-Wallis test was KW (df=1) =2.0292, 

with p=0.154, and for STAI 2 Kruskal-Wallis test 

was KW (df=1) =3.3001, with p=0.069). 

DISCUSSION 

Physical examination of TMDs showed a significant 

difference between patients and asymptomatic 

subjects at measurement of maximum mouth 

opening (14). Bates et al found a similar range of 

opening movement in the patients’ sample with 

OA of TMJ (15). 

A qualitative systematic review has shown that 

the stabilization splint is more effective in patients 

with myogenous pain than pain related to 

TMJ (3). It was shown that occlusal splint is the 

recommend management in the preprosthetic 

treatment, especially in patients with long-term 

painful TMJs with OA (16). 

Although 83% of all patients had chronic TMJ 

pain, Ekberg and Nilner (4) found that the stabilization 

splint reduced subjective symptoms of TMD. 

Ekberg et al. (5) showed that both the stabilization 

splint and controlled oral appliance had a positive 

effect on pain. Kuttila et al. (6) showed that the 

stabilization splint, like in the study of Ekberg et 

Table 2. Number and STAI test scores median values of the 

patients divided according to pain experienced before splint 

treatment and the success in pain removing* 

variables 

experienced pain 

n (%) STAI 1 STAI 2 

chronic pain 8 (50%) 45.5 43.0 

acute, subacute pain 8 (50%) 33.0 36.0 

Kruskal-Walis test 

success of splint treatment 

p=0.023 p=0.528 

painless 11 (68.75%) 36.0 37.0 

lower or unchanged pain 5 (31.25%) 43.0 49.0 

Kruskal-Walis test p=0.154 p=0.069 

*n, number of patients; STAI, State-Trait Anxiety Inventory 

al. (5), reduced clinical signs of TMD and additional 

symptoms of secondary otalgia. According to 

the results of Conti et al. (7), various modifications 

of stabilization splint improve painless condition 

in the patients with disc displacement. 

Influence of pain frequency in pre-treatment period 

as well as the participation of psychological 

factors, for example anxiety widely extended to 

general population, may have a negative influence 

on the results of TMJ pain therapy (16-19). 

There are some limitations in this study, the sample 

of patients is small and there are not many 

studies about OA of TMJ only; mainly, the study 

was conducted on various samples of TMD patients, 

which was presented in the discussion (20). 

In this study, dental students showed a statistically 

significant higher capacity of mouth opening, 

and a lower level of anxiety than patients. 

Pain duration before examination was statistically 

significant for patients: STAI 1 scores were 

higher for patients with chronic pain as compared 

to patients with acute pain. 


This study is a part of the scientific projects No. 

065-0650445-0441 and 065-0650448-0438 supported 

by the Ministry of Science, Education and 

Sports, Republic of Croatia. 

REFERENCES 

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disorders of the temporomandibular joint; etiology, 

diagnosis, and treatment. J Dent Res 2008; 87:296- 

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2. Lewis EL, Dolwick MF, Abramowicz S, Reeder SL. 

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3. Türp JC, Komine F, Hugger A. Efficacy of stablization 

splints for the management of patients with 

masticatory muscle pain: a quantitative systematic 

review. Clin Oral Invest 2004; 8:179-95. 

4. Ekberg E, Nilner M. A 6- and 12- month follow-up 

of appliance therapy in TMD patients: A Follow-up 

of a Controlled Trial. Int J Prosthodont 2002; 15: 

564-70. 

5. Ekberg E, Vallon D, Nilner M. Occlusal appliance 

therapy in patients with temporomandibular disorders. 

Acta Odontol Scand 1998; 56: 122-8. 

6. Kuttila M, Le Bell Y, Savolainen-Niemi E, Kuttila S, 

Alanen P. Efficiency of occlusal appliance therapy in 

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Acta Odontol Scand 2002; 60:248-54. 

7. Conti PC, dos Santos CN, Kogawa EM, de Castro 

Ferreira Conti AC, de Araujo Cdos R. The treatment 

of painful temporomandibular joint clicking with 

oral splints: a randomized clinical trial. Am Dent

Assoc 2006; 137:1108-14. 

8. Dworkin SF, LeResche L. Research diagnostic criteria 

for temporomandibular disorders: Review, criteria, 

examinations and specifications, critique. J Craniomandib 

Disord Facial Oral Pain 1992; 6:301-55. 

9. Bummann A, Lotzmann U. TMJ disorders and orofacial 

pain - the role of dentistry in a multidisciplinary 

diagnostic approach. Stuttgart - New York: 

Thieme, 2002. 

10. Badel T, Pandurić J, Marotti M, Kern J, Krolo I. Metrička 

analiza temporomandibularnog zgloba magnetskom 

rezonancijom u asimptomatskih ispitanika 

Acta Med Croat 2008; 62:455-60. 

11. Badel T, Marotti M, Kraljević Šimunković S, Keros 

J, Kern J, Krolo I. Radiological characteristics 

of osteoarthritis of temporomandibular joint without 

disc displacement. Period Biol 2009; 111: 289-92. 

12. Spielberger CD. Priručnik za upitnik anksioznosti 

kao stanja i osobine ličnosti (STAI) (Oblik Y). Jastrebarsko: 

Naklada Slap, 2000. [croatian edition] 

13. Badel T, Pandurić J, Perenčević K. Vertikalisationsschiene 

als Initialtherapie bei totalprothetischer Versorgung. 

Quintessenz Zahntech 2002; 28: 748-56. 

14. Ćelić R, Jerolimov J, Knezovic Zlatarić, D. Relationship 

of slightly limited mandibular movements 

to temporomandibular disorders Braz Dent J 2004; 

15:151-4. 

15. Bates RE, Gremillion HA, Stewart CM. Degenerative 

joint disease. Part II: symptoms and examination 

findings. J Craniomandib Pract 1994; 12:88-92. 

16. Badel T, Pandurić J, Marotti M, Kocijan Lovko S, 

Krolo I. Inicijalna terapija osteoartritisa čeljusnoga 

zgloba. Reumatizam 2006; 53:29-32. 

17. Manfredini D, Landi N, Bandettini Di Poggio A, 

Dell’Osso L, Bosco M. A critical review on the 

importance of psychological factors in temporomandibular 

disorders. Minerva Stomatol 2003; 52: 

321-30. 

18. Sher L. Etiology and pathogenesis of anxiety disorders. 

Med Hypothesis 2001; 57:101-2. 

19. Dworkin SF. Psychological and Psychosocial Assessment. 

In: Laskin DM, Green CS, Hylander WL, 

eds. Temporomandibular disorders. An Evidence- 

Based Approach to Diagnosis and Treatment. Chicago: 

Quintessence, 2006:203-17. 

20. Martínez-Blanco M, Bagán JV, Fons A, Poveda-Roda 

R. Osteoartrosis of the temporomandibular joint. 

A clinical and radiological study of 16 patients. Med 

Oral 2004; 9:106-15. 

CASE REPORT 

Hronični epiduralni hematom dijagnosticiran 

kao meningeom 

Hakija Bečulić 1 , Rasim Skomorac 1 , Aldin Jusić 1 , 

Alma Mekić-Abazović 2 , Alma Bajtarević 3 

1 Služba za neurohirurgiju, 2 Služba za onkologiju, 3 Služba za radi- 

ologiju; Kantonalna bolnica Zenica, Zenica, Bosna i Hercegovina 

Corresponding author: Hakija Bečulić, Služba za neurohirurgiju, 

Kantonalna bolnica Zenica, Crkvice 67, 72 000 Zenica, Bosna i 

Hercegovina, Phone: +387 32 405 133; +387 32 405 534; 

E-mail: dr_beculichakija@hotmail.com 

Originalna prijava: 01. april 2010.; Korigirana verzija: 16. april 

2010.; Prihvaćeno: 19. april 2010. 


SAŽETAK 

U radu je prezentiran rijedak slučaj organiziranog 

hroničnog epiduralnog hematoma koji je 

imitirao meningeom. Pacijent je primljen na 

Službu za neurologiju Kantonalne bolnice u Zenici 

zbog gubitka svijesti i desnostrane hemipareze. 

Nativna kompjuterizirana tomografija (CT) 

pokazala je ekspanzivni intrakranijalni proces lijevo 

parijetalno, koji je radiološki dijagnosticiran 

kao meningeom. Intraoperativno ustanovljena je 

linearna fraktura lobanje i organizirani hronični 

epiduralni hematom. 

Ključne riječi: hronični epiduralni hematom, 

meningeom, kompjuterizirana tomografija 

UVOD 

Case report 

Epiduralni hematom predstavlja krvarenje između 

kosti i tvrde moždanice, obično uzrokovano 

lezijom srednje meningealne arterije, često udružen 

s linearnom frakturom kosti lobanje (1). Klasična 

manifestacija epiduralnog hematoma jeste 

inicijalni gubitak svijesti, nakon kojeg slijedi lucidni 

interval koji traje nekoliko trenutaka ili nekoliko 

dana do nekoliko sedmica ili pak godina. 

Za vrijeme lucidnog perioda raste intrakranijalni 

pritisak (2). Akutni epiduralni hematom nastaje 

kao posljedica arterijskog, dok hronični hematom 

kao posljedica venskog krvarenja. Epiduralni 

hematom javlja se u 2% svih povreda glave, od 

čega hronični epiduralni hematom u oko 6-12% 

slučajeva (2, 3). 

Obično se javlja kod mlađih pacijenata i rijedak 

je nakon 50 godina života (3). Kod hroničnog 

63

64 


epiduralnog hematoma lucidni interval traje duže 

od 13 dana (4). Bradley smatra, na osnovu razgradnje 

hemoglobinskih produkata na snimcima 

magnetne rezonance (MRI) u T1 i T2 vremenu, 

da se hronični epiduralni hematom dijagnosticira 

14 dana nakon povrede (5). 

PRIKAZ SLUČAJA 

Pedesetjednogodišnji pacijent, hronični alkoholičar, 

primljen je na Službu za neurologiju Kantonalne 

bolnice u Zenici zbog gubitka svijesti 

za vrijeme boravka u kupatilu gdje ga je istog 

dana pronašao brat. Pacijent je kod prijema bio 

svjestan, u ležećem položaju zbog opšte tjelesne 

slabosti, urednog stanja svijesti, komunikativan, 

s desnostranom hemiparezom. Pregledom nisu 

uočeni lokalni znaci povrede glave. Heteroanamnestički 

je dobijen podatak da pacijent već 

dvije godine ima slabost u desnoj ruci i nozi uz 

povremene glavobolje. 

Urađena je nativna kompjuterizirana tomografija 

(CT) glave koja je pokazala dvije hiperdenzne 

promjene: desno temporalno promjena denziteta 

79 Hounsfieldovih jedinica (HU), promjera 15 x 

12 mm, koja je odgovarala manjem meningeomu 

i lijevo parijetalno promjena istih radioloških 

karakteristika, promjera 90 x 70 mm, koja je imponovala 

kao veći meningeom. Promjena lijevo 

parijetalno djelovala je kompresivno na lijevu 

bočnu moždanu komoru i mediosagitalne strukture 

(slika 1; slika 2). Iz tehničkih razloga nije 

rađen CT snimak mozga s kontrastom. 

Nakon pregleda neurohirurga, jedanaest dana 

nakon prijema u bolnicu, indiciran je operativni 

Slika 1. Nativni CT snimak glave, aksijalni presjek: hiperdenzna 

ekspanzivna promjena lijevo parijetalno (Služba za 

neurohirurgiju, Kantonalna bolnica Zenica, 2009.) 

zahvat u općoj anesteziji. Osam dana kasnije, uz 

pristanak članova porodice, pacijent je podvrgnut 

operativnom zahvatu. 

Intraoperativno, nakon odizanja kože i periosta, 

uočena je linearna fraktura lijevo temporalno, 

koja nije bila uočljiva na CT snimku glave. 

Načinjena su tri trepanska otvora i uz pomoć 

kraniotoma odignut je koštani flap pri čemu je 

prikazan obiman epiduralni hematom, debljine 

do 4 cm u središnjem dijelu. Periferni dio hematoma 

većim dijelom bio je organiziran vezivnim 

tkivom i srastao za duru, uz formiranje vidljive 

kapsule. Hematom je u cijelosti odstranjen, kao 

i kapsula koja je pažljivim prepariranjem odvojena 

od dure. Dura je rubno suspendirana, nakon 

čega je postavljen koštani flap. Pacijent je prvog 

postoperativnog dana bio samostalno pokretan, 

urednog stanja svijesti, vitalno i hemodinamski 

stabilan. 

Na kontrolnom pregledu, mjesec dana nakon 

operacije, pacijent je bio urednog stanja svijesti 

i neurološkog nalaza. 

U radu je prikazan pacijent, hronični alkoholičar, 

često izložen padovima, kod kojeg se hronični 

epiduralni hematom manifestirao gubitkom svijesti. 

Pri inicijalnom pregledu nisu nađeni lokalni 

znaci povrede glave, ali na osnovi anamnestičkih 

podataka i izgledu hematoma, povreda se desila 

ranije (najmanje nekoliko mjeseci). Hematom je 

većim dijelom bio organiziran, što je na nativnim 

CT snimcima davalo gustoću mehkog tkiva i imponovalo 

kao konveksitetni meningeom. Fraktura 

lobanje, u fazi zarastanja, nije uočena CT 

skeniranjem. 

Akutni epiduralni hematom jeste urgentno stanje 

u neurohirurgiji i zahtijeva hitnu operativnu 

Slika 2. Nativni CT snimak glave, koronalna rekonstrukcija: 

hiperdenzna ekspanzivna promjena lijevo parijetalno, koja 

se ponaša kompresivno na mozak i bočnu moždanu komoru 

(Služba za neurohirurgiju, Kantonalna bolnica Zenica, 2009.)

intervenciju (1). Hronični epiduralni hematom 

manje je poznat klinički entitet i rjeđe se susreće 

(4). Ima sporiji klinički tok i manifestuje se konstantnom 

glavoboljom, poremećajem memorije, 

kontralateralnom slabošću ekstremiteta i poremećajem 

senzibiliteta (2). 

U diferencijalnoj dijagnozi hroničnog epiduralnog 

hematoma, između ostalog, potrebno je misliti 

i na meningeom. Oko dvije trećine meningeoma 

na nativnim CT snimcima hiperdenzni su 

zbog hipercelularnosti i prisutnih kalcifikata (6, 

7), a nakon aplikacije kontrasta dobro primaju 

kontrastno sredstvo. Ekstraaksijalne hemoragije, 

gdje spada i epiduralno krvarenje, također su hiperdenzne 

na nativnim CT snimcima, ali ne primaju 

kontrastno sredstvo (8, 9). 

Hronični epiduralni hematom je rijedak klinički 

entitet i javlja se u 6-12% slučajeva svih epiduralnih 

hematoma (2, 3). U literaturi su poznati 

slučajevi i kalcificiranih hroničnih epiduralnih 

hematoma (8) kod kojih se dijagnoza postavlja 

na osnovu CT skeniranja, duralnih metastaza dijagnosticiranih 

kao hronični epiduralni hematom 

(3), ili slučaj organiziranog hroničnog subduralnog 

hematoma koji je MRI (Magnetic Resonance 

Imaging) skeniranjem imitirao mehkotkivnu 

tumorsku masu (6), a intraoperativno je dokazan 

hronični subduralni hematom. 

Opisani slučaj ima prvenstveno dijagnostički 

značaj. Detaljna dijagnostička evaluacija pacijenta 

nije urađena iz tehničkih razloga, a što bi 

možda omogućilo bolje diferenciranje opisane 

lezije i postavljanje sumnje na epiduralnu kolekciju, 

iako je operativno liječenje bilo neupitno u 

oba slučaja. 

Iz prezentiranog slučaja i pregleda sličnih slučajeva 

u literaturi može se zaključiti da postoje 

rijetki klinički entiteti koji nameću obavezu detaljne 

dijagnostičke evaluacije svake promjene u 

intrakranijalnom prostoru. 



postoji. 

LITERATURA 

1. 

2. 

Agrawal A, Agrawal CS, Anaud K, Shailesh A. Outcome 

of traumatic extradural haematoma managed 

surgically: our experience. NJOT 2007; 6:74-6. 

Kenneth PB, Richard DH. Chronic Extradural Haematoma: 

Case report. Neurosurgery 1979; 4: 60-62. 

3. 

4. 

5. 

6. 

7. 

8. 

9. 

Muhammad SS, Muhammad EB, Syed AE. Dural 

metastases presenting as extradural hematoma: a rare 

presentation. J Pak Med Assoc 2005; 55:509-10. 

Merih IS, Aytic CAN, Mehmet HA. Chronic Supraand 

Infratentorial Epidural Hematoma: Case Report. 

Turkish Neurosurgery 2006; 16: 212-213. 

Bradley WG Jr. Hemorrhage and hemorrhagic infections 

in the brain. Neuroimaging Clin N Am 1994; 

4:707-32. 

Do-Kwon Y, Yoon-Kyung S, Jaechan P. Tumor-Like 

Presentation of Organized Chronic Subdural Hematoma. 

J. Korean Neurosurg Soc 2006; 40:199-201. 

Joung HL. Meningiomas: Diagnosis, Treatment and 

Outcome. London: Springer, 2008. 

Sumit SM, Sachin B. Chronic calcified extradural 

hematoma in a child: Case report and review of literature. 

IJNT 2008; l5:51-2. 

David NR, Ruth GR, Kizhanatham SV et all.: Extradural 

Lymphoma Presenting as an Surgical Emergency. 

Neurosurgery 1987; 20:788-90. 

Chronic epidural haematoma mimicking 

meningioma 

Hakija Bečulić1 , Rasim Skomorac1 , Aldin Jusić1 , 

Alma Mekić-Abazović2 , Alma Bajtarević3 1 2 3 Department of Neurosurgery, Department of Oncology, Department 

of Radiology; Cantonal Hospital Zenica, Zenica, Bosnia 

and Herzegovina 

ABSTRACT 

Case report 

The study presents a rare case of organised chronic 

epidural haematoma that imitated a meningioma. 

A patient was admitted to the Department of 

Neurology of the Cantonal Hospital Zenica due 

to loss of consciousness and right hemiparesis. 

Non-contrast Computed Tomography (CT) scan 

had shown an expansive intracranial process in 

the left parietal region which was radiologically 

diagnosed as a meningioma. During the operation 

a linear skull fracture and organised chronic 

epidural haematoma were found. 

Key words: chronic epidural haematoma, meningioma, 

computed tomography. 

Original submission: 01 April 2010; Revised submission: 16 

April 2010; Accepted: 19 April 2010. 

65

66 


CASE REPORT 

Placenta previa percreta with bladder 

invasion 

Siniša Šijanović1 , Mirjana Rubin1 , Zlatko 

Topolovec1 , Domagoj Vidosavljević2 , Ivanka 

Šijanović3 1Department of Gynecology and Obstetrics, University Hospital 

Osijek, 2Department of Gynecology and Obstetrics, Vukovar 

General Hospital, Vukovar, 3Vukovar General Hospital, Vukovar; 

Croatia 


Siniša Šijanović, 

Vijenac Ivana Česmičkog 12, 

31000 Osijek, Croatia 

Phone: +385 31 512 302; fax.: +385 31 512 234; 

E-mail: d-vidosavljevic@inet.hr 

Original submission: 10 May 2010; Revised submission: 09 

June 2010; Accepted: 18 June 2010. 


ABSTRACT 

A 43- year old woman, with ten previous deliveries 

and history of two cesarean sections was admitted 

to our Department at 32 weeks of gestation 

with massive vaginal hemorrhage from an ultrasound 

diagnosed placenta previa. An emergency 

cesarean section with vertical abdominal incision 

was performed. A healthy 2300 g female infant 

was delivered. Attempts to manually remove the 

placenta caused massive hemorrhage. The lower 

uterine segment was widened due to placenta 

previa with suspicious placental invasion of the 

posterior wall of the bladder. Persistent hemorrhage 

demanded bilateral anterior internal iliac 

artery ligation and suture ligation of the bleeding 

vessels with supracervical hysterectomy done. 

Key words: placenta previa percreta, bladder invasion, 

hysterectomy 

INTRODUCTION 

The placental trophoblastic tissue does not normally 

invade the myometrium. Myometrium and 

placenta are separated by the decidual plate, which 

is the normal plane of cleavage at placental 

separation after birth (1). If the placental villi 

invade the myometrium, the placenta is termed 

accrete, and when the villi invade the myometrium 

to the serosa, the placenta is termed increta. 

The penetration of the villi beyond the uterine 

wall and through the serosa is termed placenta 

percreta, and placenta accreta has been attributed 

to complete or partial absence of the decidua basilis 

(2). The relative deficiency of the decidual 

plate in the lower uterine segment may explain 

the frequent coexistence of adherent placenta and 

placenta previa (1). 

Fox identified multigravidity, previous caesarean 

section and endometrial curettage as main risk factors 

for placental adherence (3). The risk for placenta 

accreta with placenta previa is increased with 

the number of previous caesarean sections (5,6). 

Although rare, placenta percreta with bladder involvement 

represents a possible catastrophic event 

(4). The reported maternal and perinatal mortality 

rate is 20% and 30%, respectively (9). Profuse 

hemorrhage is a common manifestation and it is 

responsible for the high maternal morbidity and 

mortality rates associated with this condition (9). 

This paper presents a case of placenta previa percreta 

with bladder invasion to highlight the dangerous 

nature of this clinical entity and discuss 

the surgical management (1,9). Our case demonstrated 

that clinical impression during surgery 

may underestimate the extent of collateral blood 

supply and therefore the difficulty of surgery. Without 

the advantage of having all necessary equipment, 

personnel and blood products immediately 

available, we believe that this patient would 

have died (10). 

CASE REPORT 

A 43-year-old woman, gravida 11, para 10, with a 

history of two caesarean sections was admitted to 

our department at 32 weeks of gestation with massive 

vaginal hemorrhage from a sonographically 

diagnosed placenta previa. Fetal heart reaction 

was positive. Prenatally she had only one pelvic 

examination and ultrasound control. 

An emergency caesarean section with vertical abdominal 

incision was performed. A healthy 2300 

g female infant was delivered. Attempts to manually 

remove the placenta caused massive hemorrhage. 

The lower uterine segment was widened 

due to placenta previa with suspicious placental 

invasion of the posterior wall of the bladder. It 

was later histologically confirmed. Since haemostatic 

sutures did not control the bleeding, supracervical 

hysterectomy was performed. Attempts 

to mobilize the bladder were unsuccessful and

cystotomy revealed involvement of the bladder 

base and urologic consultation was obtained. 

Attempts to dissect the bladder from the lower 

uterine segment and resection of the bladder were 

unsuccessful because of massive hemorrhage. 

The bladder defect was repaired in two layers 

after debridement of the irregular ragged edges 

and consultations with abdominal surgeon were 

done. Persistent hemorrhage necessitated bilateral 

anterior internal iliac artery ligation and suture 

ligation of the bleeding vessels. The pelvic 

sidewalls were opened and the hypogastic arteries 

ligated to stop the hemorrhage. Penrose pelvic 

drains were placed in the Douglas space and 

the abdominal wall was closed. Estimated blood 

loss for the procedure was 6,000 ml and intraoperative 

fluid replacement consisted of 20 units 

of packed red blood cells, 6 units of cryoprecipitate, 

6 units of fresh frozen plasma and 15 liters 

of crystalloid solutions. Postoperative course was 

complicated by anemia, dilutional coagulopathy, 

hypoalbuminemia, hypoproteinemia, hypocalcemia, 

hypocalemia, pleural effusion and two 

respiratory arrests. The patient was transferred to 

the ward from the intensive care unit on 10th day 

and later discharged from hospital on 25th post 

operative day . 

Placenta previa percreta with bladder invasion is 

a rare and potentially catastrophic condition. The 

risk of placenta accreta and percreta with placenta 

previa is dependent on the number of previous 

caesarean sections (6). Some authors found that 

the risk of placenta accreta was 5% in patients 

with placenta previa and an unscarred uterus, but 

increased to 67% in patients with four or more 

previous caesarean deliveries (10). In the cases of 

placenta percreta, profuse bleeding is responsible 

for the maternal morbidity and mortality (11). It 

is often impossible to remove the placenta completely 

after delivery without “en block” resection 

of the uterus and eventually adherent organs 

if extra uterine extension is present (12). When 

placenta percreta involves the bladder, bleeding 

might be extreme and surgery is hazardous (10) 

Intraoperative bleeding is the major threat at the 

time of hysterectomy. The rate of hemorrhage 

can usually be decreased by bilateral anterior 

internal iliac artery and/or uterine artery ligation 

with hysterectomy performing the posterior 

approach by dividing the uterosacral ligaments 

and entering the vagina posteriorly. The involved 

portion of the urinary bladder is then resected 

with the hysterectomy specimen (1,9,10). In addition, 

there is a high risk of surgical morbidity, 

including hysterectomy, inadvertent bowel, bladder 

and ureteral injury and continued bleeding 

requiring exploration in about 7% of patients (9). 

Other important complications are disseminated 

intravascular coagulation (13), fistula formation 

, ureteral stricture , urinary stasis (4),infection , 

pelvic and renal abscess formation , renal compromise 

(14), transfusion reaction, sepsis, the 

acute respiratory distress syndrome and multiorgan 

failure (9,15,16). Placenta percreta is rarely 

diagnosed preoperatively but may produce 

signs and symptoms resulting from invasion of 

adjacent structures. Some authors have reported 

hematuria in patients with bladder invasion (7). 

Cystoscopy might be used to evaluate patients 

with hematuria at risk for placenta percreta but, 

if bladder invasion is identified cystoscopically, 

biopsy confirmation is contraindicated. The biopsy 

could result in profuse hemorrhage (7). Ultrasound 

might identify placenta previa with high 

degree of accuracy, but could not differentiate 

normal from abnormal implantation. Leaphart et 

al described a case diagnosed by ultrasonography 

with placenta percreta and bladder involvement 

at 16 weeks of gestation, and findings included 

complete placenta previa, extensive lower uterine 

segment venous lakes, loss of lower uterine 

segment myometrial echoes and nodular bladder 

wall with vascular invasion (10). 

Placenta percreta with bladder involvement is 

an obstetric emergency with high morbidity and 

mortality rate. The hallmark of successful treatment 

of placenta percreta is the control of hemorrhage, 

which frequently requires hysterectomy 

and bilateral hypogastric and/or uterine artery 

ligation. In established cases of placenta percreta 

with bladder invasion an organized multidisciplinary 

approach with proper planning, arrangement 

of blood products and intensive monitoring 

during anesthesia may decrease maternal and fetal 

morbidity and mortality. 



Case report 

67

68 


REFERENCES 

1. Abbas F, Talati J, Wasti S, Akram S, Ghaffar S, Qureshi 

R. Placenta percreta with bladder invasion as a 

cause of life threatening hemorrhage. J Urol 2000; 

164:1270- 4. 

2. Dubois J, Garel L, Gringnon A, Lemay M, Leduc 

L. Placenta percreta: ballon occlusion and embolization 

of the internal iliac arteries to reduce intraoperative 

blood losses. Am J Obstet Gynecol 1997; 

176:723-6. 

3. Fox H. Placenta accreta 1945-1969. Obstet Gynecol 

Surv 1972; 27:475-490. 

4. Washecka R, Behling A. Urologic complications of 

placenta percreta invading the urine bladder: a case 

report and review of the literature. Hawaii Med J 

2002; 61:66-9. 

5. Breen JL, Neubecker R, Gregori CA, Franklin JE. 

Placenta accreta, increta and percreta: A survey of 40 

cases. Obstet Gynecol 1977; 49:43-7. 

6. Nielsen TF, Hagberg H, Ljungblad U. Placenta previa 

and antepartum hemorrhage after previous cesarean 

section. Gynecol Obstet Invest 1989; 27:88-90. 

7. Meehan FP, Casey C, Costello JN, Connolly CE. 

Placenta previa percreta with bladder involvement. 

Obstet Gynecol Surv 1989; 44:835-40. 

8. Ochshorn A, David MP, Soferman N. Placenta previa 

accreta: A report of nine cases. Obstet Gynecol 

1969; 33:667-9. 

9. Price FV, Resnik E, Heller AK, Christopherson AW. 

Placenta previa percreta involving the urinary bladder: 

A report of two cases and rewiew of the literature. 


10. Leaphart WL, Schapiro H, Broome J, Welander CE, 

Bernstein IM. Placenta previa percreta with bladder 

invasion. Obstet Gynecol 1997; 89:834-5. 

11. Bauer ST, Bonanno C. Abnormal placentation. Semin 

Perinatol 2009; 33:88-96. 

12. Hudon L, Belfort MA, Broome DR. Diagnosis and 

management of placenta previa percreta:a review. 

Obstet Gynecol Surv 1998; 53:509-17. 

13. Luo G., Perni SC, Jean Pierre C.Baergen RN, Predanic 

M.Failure of conservative management of placenta 

previa percreta. J Perinat Med 2005; 33:564-8. 

14. Konijeti R, Rajfer J, Askari A. Placenta previa and 

the urologist. Rev Urol 2009; 11:173-6. 

15. Papp Z. Massive obstetric haemorrhage. J Perinat 

Med 2003; 31:408-14. 

16. Shevell T, Malone FD. Management of obstetrics 

haemorrhage. Semin Perinatol 2003; 27:86-104. 

CASE REPORT 

Endometrijalni karcinom povezan s 

porastom CA 15_3 i CA 125 u bolesnice 

s karcinomom dojke liječene tamoksifenom 

Alma Mekić-Abazović1 , Hakija Bečulić2 , Miralem 

Musić3 , Almir Fajkić3 , Senad Dervišević4 1 2 Služba za onkologiju i hematologiju, Služba za neurohirurgiju; 

Kantonalna bolnica Zenica, Zenica, Bosna i Hercegovina; 3Insti tut za patofiziologiju; Medicinski fakultet u Sarajevu, Sarajevo, 

Bosna i Hercegovina; 4Služba za hirurške bolesti, Kantonalna 

bolnica Zenica, Zenica, Bosna i Hercegovina 


Alma Mekić-Abazović, Služba za onkologiju i hematologiju, 

Kantonalna bolnica Zenica, Crkvice 67, 72 000 Zenica, Bosna 

i Hercegovina, Phone: +387 32 201 680; fax: +387 32 202 

681; E-mail: dralmaa@hotmail.com 

Originalna prijava: 15. juni 2010.; Korigirana verzija: 26. juli 

2010.; Prihvaćeno: 23. august 2010. 


SAŽETAK 

U radu je prezentiran slučaj endometrijalnog 

karcinoma kod pacijentice s rakom dojke tretiranim 

tamoksifenom. Bolest se manifestovala 

povišenim vrijednostima tumorskih markera CA 

125 i CA 15_3, bez drugih znakova. Urađene su 

dodatne dijagnostičke analize koje su pokazale 

da se radi o de novo nastalom endometrijalnom 

karcinomu, a ne o metastatskoj promjeni karcinoma 

dojke. Pacijentica je podvrgnuta operativnom 

zahvatu i radioterapiji. Nakon toga, vrijednosti 

tumorskih markera bile su u referentnim vrijednostima. 

Ključne riječi: karcinom dojke, tamoksifen, tumorski 

markeri, endometrijalni karcinom 

UVOD 

Rak tijela materice predstavlja jedan od najčešćih 

malignoma u ženskoj populaciji (1). Incidenca u 

razvijenim zapadnim zemljama kreće se oko deset 

novih slučajeva na stotinu hiljada žena (1, 2). 

Faktori rizika za razvoj karcinoma endometrijuma 

jesu gojaznost, rana menarha, kasna menopauza, 

anovulatorni ciklusi, postojanje hiperplastičnih 

promjena na endometrijumu i mioma na 

materici (1, 2). Hiperestrogenemija, endogena 

ili uzrokovana unošenjem egzogenih preparata,

povećava rizik od nastanka karcinoma tijela uterusa 

(1). Adenomioza, koja je prisutna kod 2-3% 

žena, povećava rizik od miometrijalne invazije 

endometrijalnog karcinoma (3, 4). U posljednje 

dvije decenije, u velikom broju istraživanja je 

dokazano da kod žena koje su zbog karcinoma 

dojke bile na terapiji tamoksifenom (selektivni 

modulator estrogenih receptora), postoji povećan 

rizik od razvoja karcinoma endometrijuma (1, 2). 

U praćenju i dijagnostičkoj evaluaciji pacijentica 

s karcinomima dojke i ženskog genitalnog sistema, 

osim drugih dijagnostičkih metoda, koriste 

se i tumorski markeri (5, 6). Posebno su korisni u 

evaluaciji odgovora na terapiju (2, 5). Inače, tumorski 

markeri nisu apsolutno specifični za vrstu 

tumora i porast njihove koncentracije može postojati 

i kod nekih benignih stanja (1, 5, 6). Zato se 

vrijednost pojedinačnog tumorskog markera, bez 

pomoći drugih dijagnostičkih metoda, ne može 

koristiti u dijagnostici tumora, ali porast njihove 

koncentracije može ukazati na pojavu rekurensa 

tumora ili pojavu metastaza (6-8). Karbohidratni 

antigen CA 15-3 koristi se u evaluaciji i praćenju 

raka dojke, ali nije strogo specifičan, te porast 

njegove koncentracije može da se detektuje i kod 

raka ovarijuma, debelog crijeva, pluća, endometrijuma, 

ali i kod drugih malignoma (3). Karbohidratni 

antigen CA-125 je specifičniji za ovarijum, 

ali se u povećanim koncentracijama može naći i 

kod raka dojke, materice, pluća, jetre, cerviksa, 

kolorektuma (3). 

U ovom radu prezentirat ćemo slučaj pacijentice 

kod koje je otkriven endometrijalni karcinom, a 

koja je prethodno liječena hormonalnom terapijom 

tamoksifenom zbog karcinoma dojke, što bi 

moglo poslužiti kao primjer za prosuđivanje u 

kliničkom radu. 

Šezdesetogodišnja pacijentica, dijabetičarka na 

inzulinskoj terapiji, prvi put se javila onkologu, 

na Službu za onkologiju Kantonalne bolnice u 

Zenici, juna 2008. godine. 

Pacijentici je 2001. godine urađena radikalna 

mastektomija lijeve dojke uslijed dijagnosticiranog 

malignog procesa. Patohistološki nalaz tada 

je pokazao da se radilo o invazivnom duktalnom 

karcinomu dojke s karcinomatoznom infiltracijom 

mišića. Maligne ćelije su imunohistohemijski 

pokazivale ekspresiju ER-a (estrogeni receptori) 

i PR-a (progesteronski receptori) na oko 

50% ćelija u srednjem intenzitetu boje, dok su 

Case report 

bile HER-2 negativne. Postoperativno pacijentica 

je bila podvrgnuta hemoterapiji (šest ciklusa) 

i terapiji ozračivanjem, a pet je godina bila na 

hormonalnoj terapiji tamoksifenom. Posljednja 

kontrolna mamografija, rađena u julu 2007. godine, 

nije pokazala znakove recidiva. 

Pacijentica je unazad deset godina bila pod kontrolom 

ginekologa zbog mioma na materici. 

Pri pregledu iz juna 2008. godine lokalni nalaz na 

dojci bio je uredan. Palpacijom je ustanovljena 

tumorska tvorba u donjem dijelu trbuha (oko 7 x 

7 cm). Kontrolni ultrazvučni pregled dojki i abdomena 

bio je uredan, kao i radiografski snimak 

pluća. Vrijednost tumorskog markera CA 15-3 

iznosila je 63.2. S obzirom na povišenu vrijednost 

CA 15-3, preporučili smo hitan pregled ginekologa, 

mamografiju i scintigrafiju skeleta, te nalaz 

CA 125 i kontrolni CA 15-3. Scintigrafija skeleta 

(PRISM 2000xP, Cleveland, USA) je pokazala 

promjene artritičnog tipa, a mamografija nije 

pokazala znakove recidiva bolesti. Ginekološkim 

ultrazvučnim pregledom (Sonoace, Medison Co. 

ltd. Korea) opisan je miom na stražnjem zidu 

materice, promjera 60 x 65 mm, endometrijum 

debljine 4 mm bez patoloških promjena. Ista je 

promjena opisivana i pri prethodnim ginekološkim 

ultrazvučnim pregledima (bez progresije u 

veličini). Color Doppler i transvaginalni ultrazvuk 

nisu rađeni iz tehničkih razloga. 

Na kontrolnom pregledu onkologa, dvije sedmice 

nakon prethodnog, urađeni su tumorski markeri, 

a koji su prethodno indicirani. Laboratorijski nalazi 

su pokazali slijedeće vrijednosti tumorskih 

markera: CA 125 je iznosio 43.4. a CA 15_3 

iznosio je 62.2. 

S obzirom na porast navedenih tumorskih markera 

koji su sugerisali da se moglo raditi o malignitetu 

u predjelu ženskog genitalnog sistema, 

u dogovoru s ginekologom, pacijentica je podvrgnuta 

operativnom zahvatu. Uz pisani pristanak 

pacijentice urađena je histerektomija s obostranom 

adneksektomijom. Prethodno nije rađena 

frakcionirana kiretaža jer se radilo o ultrazvučno 

verificiranom miomu velikih dimenzija, bez vidljivih 

patoloških promjena na endometrijumu. 

Kompletan odstranjeni sadržaj poslan je na patohistološku 

analizu. Patohistološkim pregledom 

je tumorski čvor promjera 10 cm u zidu 

tijela materice, dijagnosticiran kao intramuralni 

lejomiom, a tumorski čvor promjera 3 cm u mi- 

69

70 


šićnom sloju, kao žarište mucinoznog intramuralnog 

adenokarcinoma, dok u endometrijumu 

nisu nađene patološke promjene (promjena se 

nalazila u miometrijumu i nije bila u kontaktu s 

endometrijem). Pacijentici je, s obzirom na veličinu 

i lokalizaciju tumora, kao i stadijum bolesti, 

predložena radioterapija koja je sprovedena bez 

komplikacija. Kontrolni tumorski markeri, koji 

su učinjeni nakon operacije i provedene radioterapije, 

bili su u referentnim vrijednostima. Vrijednost 

CA 125 je iznosila 4.9. a vrijednost CA 

15_3 iznosila je 6.3. 

U prezentiranom slučaju, kontrolnom analizom 

tumorskih markera CA 15_3 i CA 125, kod pacijentice, 

koja je prethodno bila liječena od karcinoma 

dojke, postavljena je sumnja na postojanje 

maligniteta u predjelu ženskog genitalnog 

sistema, što je i potvrdila patohistološka analiza 

operativno odstranjenih unutrašnjih genitalnih 

organa pacijentice. Radilo se o de novo nastalom 

endometrijalnom karcinomu, a ne o metastatskoj 

promjeni, što se moglo očekivati s obzirom na 

osnovnu bolest (karcinom dojke). 

Jasan dokaz za ovu tvrdnju bio je i histološki nalaz 

endometrijalnog karcinoma koji je potvrdio 

da se radilo o adenokarcinomu, za razliku od karcinoma 

dojke koji je bio duktalnog porijekla. 

Postojanje intaktnog endometrijuma sugerirao 

je da se karcinom razvio u žarištu adenomioze, 

što, prema podacima iz literature, povećava rizik 

od invazivnog ponašanja tumora (4). Osim 

toga, pacijentica je već pet godina bila na terapiji 

tamoksifenom što, prema istraživanjima, značajno 

povećava rizik od razvoja endometrijalnog 

karcinoma. Naime, istraživanja pokazuju da se 

endometrijalni karcinom javlja kod 1,6 do 2,0 

slučajeva na 1.000 pacijentica tretiranih tamoksifenom 

(1, 2). 

U praćenju i evaluaciji odgovora na terapiju kod 

karcinoma dojke koristi se karbohidratni antigen 

CA 15-3, dok se kod karcinoma na ženskom genitalnom 

sistemu koristi karbohidratni antigen CA- 

125 (5). Istraživanja su pokazala da vrijednost 

CA 125 kod endometrijalnog karcinoma korelira 

s dubinom invazije miometrijuma, stadijumom 

bolesti, invazijom cerviksa, citološkim statusom, 

te statusom regionalnih limfnih čvorova i prognozom 

(6). Vrijednost CA 125 iznad 35 umol/l 

sugerira s velikom vjerovatnoćom ekstrauterino 

širenje bolesti, dok vrijednost iznad 20 umol/l 

kod 75% do 87% slučajeva ukazuje na zahvaćenost 

regionalnih limfnih čvorova (7, 8). U našem 

primjeru bolest ne samo da nije detektovana na 

ekstrauterinim strukturama, nego nije bila vidljiva 

ni ultrazvučnim pregledom. Tumorski markeri 

CA 125 i CA 15_3 bili su jedini parametri koji 

su konstantno bili povišeni. Kontrolni marker CA 

15_3 urađen je nakon dvije nedjelje u našoj ustanovi, 

jer je inicijalni CA 15_3 s kojim je pacijentica 

došla na kontrolu urađen ambulantno. Uobičajeno 

je da se tumorski markeri rade svakih 6 do 

12 mjeseci ovisno od kliničkog i lokalnog nalaza, 

te suspektnih dijagnostičkih procedura. 

Kod 32% slučajeva endometrijalnog karcinoma 

ustanovljen je porast CA 15-3, dok je kod 87% 

korelirao s terapijskim odgovorom (9). I u našem 

slučaju, nakon odstranjenja tumora, došlo je do 

normalizacije vrijednosti CA 125, ali i CA 15_3. 

Iz prezentiranog slučaja može se zaključiti da tumorski 

markeri imaju veliki značaj u postoperativnom 

praćenju bolesti, ali isto tako i da njihov 

porast ne mora značiti samo pojavu metastatskih 

promjena ili lokalnog recidiva, nego i pojavu novog 

maligniteta, na što se često zaboravi tokom 

praćenja pacijenta. Svaki porast tumorskih markera 

zahtijeva detaljnu dijagnostičku evaluaciju 

pacijenta. Ordinirajući onkolog treba se fokusirati 

ne samo na praćenje oboljelog organa, nego na 

organizam u cjelini. 

Prikazani bi slučaj mogao biti osnova ljekarima 

na tercijarnom nivou zdravstvene zaštite za jedan 

detaljniji i širi dijagnostički pristup onkološkom 

pacijentu. 



postoji. 

LITERATURA 

1. Emons G, Fleckenstein G, Hinney B, Huschmand 

A, Heyl W. Hormonal interactions in endometrial 

cancer. Endocrine-Related Cancer 2000; 7:227-42. 

2. 

Barakat RR, Gilewski TA, Almadrones L, Saigo PE, 

Venkatraman E, Hudis C, Hoskins WJ. Effects of 

Adjuvant tamoxifen on the endometrium in women 

with breast cancer: a prospective study using office 

endometrial biopsy. J Clin Oncol 2000; 18:3459-63. 

3. Gara S, Boussen H, Ghanem A, Guemira F. Use of 

4. 

common seric tumor markers in patients with solid 

cancers. Tunis Med 2008; 86:579-83. 

Monica S, Walter K, Robert K. CD10 Immunosta- 

ining does not distinguish endometrial carcinoma 

invading myometrium from carcinoma involving

adenomyosis. Am J Surg Pathol 2003; 27:786-9. 

5. Gadducci A, Cosio S, Carpi A, Nicolini A, Genazzani 

AR. Serum tumor markers in the management 

of ovarian, endometrial and cervical cancer. Biomed 

Pharmacother 2004; 58:24-38. 

6. Deborah JD. 

Preoperative CA 125 in endometrial 

cancer: Is it useful? Am J Obstet Gynecol 2000; 

182:1328-34. 

7. Peter K, Yedema CA, Bon GG, and Silvia von MP. 

CA 125 in gynecological pathology - a review. Eur J 

Obstet Gynecol Reprod Biol 1993; 49:115-24. 

8. Seoud M, Joanna J, John W. Gynecologic tumors in 

tamoxifen-treated women with breast cancer. Obstet 

Gynecol. 1993; 82:165-9. 

9. Scambia G, Benedetti Panici P, Baiocchi G, Perrone 

L, Greggi S, Mancuso S. CA 15-3 as a tumor marker 

in gynecological malignancies. Gynecol Oncol 

1988; 30:265-73. 

Endometrial cancer associated with 

an increase of CA 15_3 and CA 125 in 

tamoxifen treated patients with breast 

cancer 

Alma Mekić-Abazović1 , Hakija Bečulić2 , Miralem 

Musić3 , Almir Fajkić3 , Senad Dervišević4 1 2 Department of Oncology and Haematology, Department of 

Neurosurgery; 

Cantonal Hospital Zenica, Zenica, Bosnia and Herzegovina, 

3Institute of Pathophysiology, School of Medicine, University 

of Sarajevo, Sarajevo, Bosnia and Herzegovina, 4Department 

of Surgery, Cantonal Hospital Zenica, Zenica, Bosnia and 

Herzegovina 

ABSTRACT 

The study presents a case of endometrial cancer 

in a breast cancer patient treated with tamoxifen. 

The disease occured with elevated values of 

CA 125 and CA 15_3 tumour markers without 

any other signs. Additional diagnostic analyses 

were performed showing a „de novo“ endometrial 

cancer rather than metastatic breast cancer. 

The patient underwent surgery and radiotherapy. 

Thereafter, the values of tumour markers were in 

the reference values. 

Key words: breast cancer, tamoxifen, tumour 

markers, endometrial carcinoma 

Original submission: 15 June 2010; Revised submission: 26 

July 2010; Accepted: 23 August 2010. 

CASE REPORT 

Primary malignant melanoma of gallbladder 

Vlatka Pitlović, Ferid Latić, Hrvoje Pitlović, Mario 

Rupčić, Darko Jurišić, Azra Latić 

Department of Surgery, General Hospital “Dr. Josip Benčević”, 

Slavonski Brod, Croatia 


Vlatka Pitlović, Department of Surgery, General Hospital “Dr. 

Josip Benčević”, Andrije Štampara 42, 35 000 Slavonski Brod, 

Croatia, Phone : +385 35 201 653, E-mail: vpit@net.hr 

Original submission: 07 January 2010; Revised submission: 

21 March 2010; Accepted: 08 April 2010. 


ABSTRACT 

Gallbladder primary malignant melanoma 

(GPMM) is a rare and controversial entity. The 

existing controversy on the subject appears mainly 

because of the lack of definitive objective criteria 

of primitivity. Objective criteria proposed by the 

specific literature for distinguishing GPMM from 

secondary gallbladder melanoma include the exclusion 

of previous primitive melanoma, absence of 

synchronous involment of other sites, the unicity 

of lesion, its polipoid or papilary shape and the 

presence of a junctional melanocitary component. 

After laparoscopic cholecystectomy in one of our 

patients, dark polypus inside the gallbladder was 

found. A malignant melanoma was diagnosed 

according to all five criteria for GPMM. 

Key words: gallbladder, primary malignant melanoma, 

laparoscopic cholecystectomy 

INTRODUCTION 

Case report 

Melanoma arising from the mucosal epithelium 

of gallbladder was first described by Wieting and 

Hamdi in 1907 based on autopsy findings in a 

female aged 40 (1,2). The migration of melaninproducing 

cells from the neural crest to endodermal 

derivates such as the oesophageus, rectum, 

vagina and gallbladder during embryologic development 

explains and supports the possibility of 

developing primary melanoma at these sites (3). 

The most common presentation of symptomatic 

gallbladder melanoma imitates acute cholecystitis 

(4). The method of choice for diagnosis is 

ultrasound scanning (5). The laparoscopic cholecystectomy 

is indicated for patients with isolated, 

71

72 


resectible gallbladder melanoma who could achieve 

longer survival (6-10). 

In this paper we have presented a case of primary 

malignant melanoma treated by laparoscopic 

cholecystectomy. 

CASE REPORT 

A fifty-two year old man complained about right 

hypochondral pain which was relieved after spasmoanalgetics. 

Laboratory tests were normal (white blood cells, 

liver enzymes, serum amylase). 

Using ultrasound scan a polypoid mass was detected 

in the gallbladder. A laparoscopic cholecystectomy 

was performed and dark polypoid 

tumor, measuring 15x 10x 5 mm, attached to 

gallbladder neck was found. Histologycally, the 

polypoid lesion was malignant melanoma confined 

to the lamina propria with junctional activity 

(Figure 1). Many cells stained positively for 

S100 protein using immunohystochemistry and 

contained dark brown pigment that stained as 

melanin pigment . 

After the histologycal diagnosis has been made a 

search was conducted to find an alternative primary 

tumor site. This has included examination 

of the skin, proctoscopy, gastroscopy, fundoscopy, 

abdominal CT, and all of them have shown no 

abnormalities. The patient’s postoperative course 

was uneventful, but 20.5 months later brain metastasis 

was found (Figure 2). The patient died 

Figure 1. Microscopic picture of melanoma in the gallbladder 

(HE x20) (Department of Pathology, General Hospital “Dr. Josip 

Benčević“, Slavonski Brod, 2003) 

shortly after that, 22.5 months after the initial 

diagnosis of gallbladder melanoma. 

Both primary and metastatic melanoma of gallbladder 

are extremely rare (6,7). 

Distinguishing primary from secondary melanoma 

of gallbladder is an academic exercise, because 

of only one notable long-term survivor, so the 

overall prognosis is poor and the patients usually 

die of metastatic disease (5,6,8,11). 

We could conclude that the present case was probably 

gallbladder primary malignant melanoma 

(GPMM) because all of the objective criteria proposed 

by the specific literature for distinguishing 

GPMM from secondary gallbladder melanoma 

were satisfied before primary malignant melanoma 

was diagnosed: an exclusion of a previous 

primitive melanoma, absence of synchronous involvement 

of the other sites, the unicity of lesion, 

its polipoid or papilary shape and the presence of 

a junctional melanocitary component (10,11). 

The tumor was solitary and arose from mucosal 

surface of the gallbladder, it was polpoid, and 

displayed junctional activity on histological examination. 

In addition, the disease was diagnosed 

after cholecystectomy and a careful examination 

of the skin and extracutaneous sites, where primary 

melanomas usually occur (8,9), had failed 

to show any tumor deposits. 

Because of the rarity of the primary melanomas 

of gallbladder, no therapeutic guidelines could be 

recommended. Nevertheless, aggressive surgical 

Figure 2. A computed tomography scan shows metastasis in 

brain (Department of Radiology, General Hospital “Dr. Josip 

Benčević “, Slavonski Brod, 2005)

therapy prolonged survival, and improved the quality 

of life in several patients (6-10). 

In the patients who underwent cholecystectomy, 

the median survival following treatment was 16 

months, as compared to 6 months for those treated 

without surgery (7). Currently, surgical management 

appears to be indicated for patients with 

isolated, resectable gallbladder metastases (8). 

ACKNOWLEDGMENT/DISCLOCURE 

Competing interests: none declared 

REFERENCES 

1. Hamdi WH. Über di physiologische und pathologische 

melanin pigmentierung und epithelialen ursprung 

der melanoblastoma ein primares lelanoblastom der 

gallenblase. Beitr Path Anat 1907; 42:23-84. 

2. Ricci R, Maggiano N, Martini M, Antonio MA, 

Mule FP, Capelli A, Larocca LM. Primary malignant 

melanoma of the gallbladder in dysplastic naevus 

syndrome. Virchows Arch 2001; 438:159-65. 

3. Le Dourarin NM. Cell migration in embryos. Cell 

1984; 38:353-60. 

Case report 

4. Vernadakis S, Rallis G, Danias N, Costas S, Christodoulou 

E, Troullinakis M, Legakis N, Peros G. 

Metastatic melanoma of the gallbladder: an unusual 

clinical presentation of acute cholecystitis. W J Gastroenterol 

2009;15:3434-6. 

5. Goldin EG. Malignant melanoma metastatic to the 

gallbladder. Case report and review of the literature. 

Am Surg 1990; 56:369-73. 

6. Gogas J, Mantas D, Gogas H, Kouskos E, Markopoulos 

C, Vgenopoulou S. Metastatic melanoma in 

the gallbladder: report of a case. Surg Today 2003; 

33:135-7. 

7. Tuveri M, Tuveri A . Isolated metastatic melanoma 

to the gallbladder: Is laparoscopic cholecystectomy 

indicated? A case report and review of the literature. 

Surg Lap End Percut Tech 2007; 17:141-4. 

8. Dong XD, Dematos P, Prieto VG, Seigler HF. Melanoma 

of the gallbladder;a review of cases seen 

at Duke university Medical center. Cancer 1999; 

85:32-9. 

9. Katz SC, Bowne WB, Wolchok JD, Busam KJ, Jaques 

DP, Coit DG. Surgical management of melanoma 

of the gallbladder: A report of 13 case and review of 

the literature. Am J Surg 2007; 193:493-7. 

10. Heath D, Womack C. Primary malignant melanoma 

of the gallbladder. J Clin Pathol 1988; 41:1073-7. 

11. Velez AF, Penetrante RB, Spellman JE. Malignant 

melanoma of the gallbladder: report of a case and 

review of the literature. Am Surg 1995; 61:1095-8. 

73

EDITORIAL 

Medical biochemistry in everiday clinical praxis 

We have had an opportunity to read issues related to 

medical biochemistry in everyday clinical praxis. In 

any hospital and in medical praxis of primary care 

with family medicine, biochemistry is an additional 

job traditionally taken as a „medical service“ for diagnostic 

procedures, or follow up of some diseases. 

Attitude to take medical biochemistry as the „medical 

service“, which is one of the most important in 

the clinical guidelines, is the intention of the medical 

staff who consider patophysiologal pathways to be a 

cornerstone in their decision making. There is no possibility 

to understand any disease without knowledge 

of biochemical pathways in the body. Understanding 

biochemistry is undoubtedly a part of most important 

skills of excellent experience of clinicians. On the 

other hand, biochemistry is the most important field 

overlapping with different clinical services. 

As any other medical specialty medical biochemistry 

has gained a lot of knowledge in last two decades, 

mostly at the molecular level and in developing 

understanding of subcellular events during pathological 

processes. Among these, new knowledge of 

signal transduction from cellular membrane to inner 

part of the cell is most interesting for its introduction 

in everyday clinical praxis. It is up to clinical 

practitioners and their skills of understanding basic 

biological processes involved in the pathophysiological 

patterns, while progressing towards an illness, 

to decide to what extent new knowledge could be 

introduced in everyday medical practice. It is very 

important to distinguish „routine medical practice 

based on experience“ from „medical practice in 

which basic pathological processes are considered“. 

Understanding basic pathophysiological processes 

is conditio sine qua non for reasonable decision making 

in diagnostic and therapeutic procedures. 

Clinical influence of basic medical research is the field 

of high importance for any hospital and medical practitioners 

who tend to reach high level of understanding 

of their own practice. In these activities medical and 

scientific staff involved in basic research should be 

the connection between basic research and everyday 

practice. Specialists of medical biochemistry, immunologists, 

and doctors involved in medical physiology 

and pathophysiology are cornerstones of the bridges 

between basic scientific knowledge and their application 

in everyday clinical practice. One of the best possibilities 

to introduce this basic medical science and 

its use in clinical practice, is to establish departments 

of clinical pathophysiology in hospitals. In this manner 

clinical pathophysiology could be a medical service 

oriented to patients without precise diagnosis after 

many diagnostic procedures. 

This thematic issue of the Journal is done on the occasion 

of the First Congress of Medical Biochemistry, 

with international participation, which took place in 

Sarajevo in May 2010. Most of these articles have 

been done as presentations for the Congress, or they 

were an output of same researches prepared for the 

purpose of the Congress. The fields of interest of these 

articles are so different, but they have been related to 

medical biochemistry. It may be noted that the Journal 

contains articles related to different clinical pathology, 

including use of medical biochemistry for the purpose 

of follow up of different manners of treatment of 

bleeding ulcers with proton pomp inhibitors, for the 

follow up of cardiovascular comorbidities in patients 

on hemodyalsis, use of biomarkers for the follow up 

of water overload in patients with kidney diseases and 

so on. A few articles share similar methods made as 

experimental and clinical procedures, like the analysis 

of NT pro-BNP, and other markers of cardiac tissue 

damage. There are a few articles related to basic medical 

research in the fields of genetic polymorphism 

in metabolic diseases, like diabetes, and lung cancer. 

Therefore, the position of above mentioned Congress 

and this issue of the Journal is that a connection 

between medical biochemistry and everyday medical 

praxis needs to be made and results of basic medical 

research for purpose of use in everyday clinical praxis 

are to be introduced. 

On behalf of the Editorial Board of Medicinski glasnik 

we would like to thank all the authors of the 

articles provided for this issue, and all contributors 

of the First Congress of Medical Biochemistry of 

Bosnia and Herzegovina with international participation. 

Our intention is to use medical biochemistry 

not only as the additional service to clinicians, but 

also to introduce these specialists in everyday work 

in hospital departments. 

Ass. Prof. Dr Besim Prnjavorac,MD, PhD 

Guest Editor 

Prof Dr Selma Uzunović-Kamberović 

Editor-in-Chief 

Medicinski Glasnik 

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Association of PPARG and LPIN1 gene polymorphisms with 

metabolic syndrome and type 2 diabetes 

Tamer Bego 1 , Tanja Dujic 1 , Barbara Mlinar 2 , Sabina Semiz 1 , Maja Malenica 1 , Besim Prnjavorac 3,4 , Barbara 

Ostanek 2 , Janja Marc 2 , Adlija Čaušević 1 

1Department for Biochemistry and Clinical Analysis, Faculty of Pharmacy, University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 

2 3 Department for Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia, General Hospital Tešanj, 

Tešanj, 4Department of Patophysiology, Faculty of Pharmacy, University of Sarajevo, Sarajevo; Bosnia and Herzegovina 

Corresponding author 

Bego Tamer 

Department for Biochemistry and Clinical 

Analysis, Faculty of Pharmacy 

University of Sarajevo 

Čekaluša 90, 71000 Sarajevo, 


Phone: +387 33 269 640; 

fax: +387 33 269 640 

E-mail: tamer.bego@gmail.com 


22 October 2010; 


26 November 2010; 

Accepted: 

27 November 2010. 

ABSTRACT 


Aim Lipin 1 is a recently discovered multifunctional protein involved 

in the metabolism of lipids, while PPARγ is involved in adipocyte 

differentiation, and regulation of lipid metabolism. Up to 

now, LPIN1 and PPARG gene polymorphisms have been associated 

with type 2 diabetes, metabolic syndrome, and central obesity. 

In this study, we hypothesized that genetic variants within LPIN1 

and PPARG genes were associated with traits of metabolic syndrome. 

Correlation between biochemical parameters (including but 

not limited to, glucose, HbA1c, insulin levels, HDL and LDL cholesterol, 

triglycerides, serum proteins, liver enzymes) and frequency 

of polymorphisms in LPIN1 (rs11693809 and rs2716610) and 

PPARG gene (rs10865710, rs3856806 and rs1801282), was tested 

in this study. 

Methods The study included 70 patients diagnosed with metabolic 

syndrome and type 2 diabetes. Two polymorphisms of LPIN1 gene 

(rs11693809 and rs2716610), and three polymorphisms of PPARG 

gene (rs10865710, rs385806 and rs1801282) were analyzed by 

real time PCR and conventional PCR-RFLP methods. 

Results Our analysis revealed correlation between insulin levels 

and rs11693809 LPIN1 polymorphism in diabetic patients. Also 

the results of this study showed an association of rs10865710 and 

rs385806 polymorphism of PPARG with HDL cholesterol and 

LDL plus total cholesterol levels, respectively. 

Conclusion These data reflect an association of analyzed PPARG 

and LPIN1 gene polymorphisms with values of insulin, HDL, 

LDL and total cholesterol witch indicates an important role of these 

genes in lipid metabolism and pathogenesis of type 2 diabetes 

and metabolic syndrome. 

Key words: metabolic syndrome, LPIN1, PPARG, type 2 diabetes.

INTRODUCTION 

Metabolic syndrome is the common name for a 

cluster of metabolic disorders that together represent 

an indicator of the risk for diabetes, heart disease, 

stroke and other cardiovascular conditions. Its 

metabolic risk factors are insulin resistance, abdominal 

obesity, hypertension, and dyslipidemia (1). 

The American organization NCEP (National Cholesterol 

Education Program) defines how to properly 

diagnose metabolic syndrome in a patient. 

Three or more of the following criteria have to be 

met: fasting glucose ≥ 6.1 mmol / l, serum triglycerides 

≥ 1.7 mmol / l, serum HDL cholesterol < 

1.4 mmol / l in men or < 1.29 mmol / l in women, 

blood pressure ≥ 130/85 mm Hg, waist circumference 

> 102 cm in men or > 88 cm in women (2). 

Definition of the metabolic syndrome by IDF 

(International Diabetes Federation) includes the 

presence of abdominal obesity in a patient with 

two or more NCEP criteria fulfilled, with a lower 

limit for fasting glucose (5.6 mmol / l) (3). 

The pathogenesis of the metabolic syndrome is 

thought to involve a complex interaction of multiple 

factors, which include obesity and abnormal 

fat distribution, insulin resistance, hepatic, vascular, 

and immunologic factors, and lifestyle and 

genetic contributions (4-6). 

Obesity is a contributing factor to hypertension, 

high cholesterol levels, low HDL cholesterol, and 

hyperglycemia, which are jointly associated with 

a high risk of cardiovascular diseases. Excess fat 

is responsible for an increase of non-esterified fatty 

acids, which overloads muscles and liver, and 

leads to the development of insulin resistance. 

Insulin resistance or hyperinsulinemia, directly 

affects some risks factors for metabolic syndrome, 

and generally correlates with the increase of 

body fat (7, 8). 

Heritability of the metabolic syndrome, according 

to NCEP criteria, is estimated to be around 

30% (9). It is specific for certain populations 

and generally reflects the diversity of population 

tested and its environment (10, 11). Data related 

to the prevalence of metabolic syndrome in the 

group of 60-69 years of age, show a different representation 

of the incidence of metabolic syndrome 

in different populations (the incidence of 

metabolic syndrome in the USA is 42%, while in 

France it is 20%) (12,13). 

Bego et al PPARG and LPIN1 genes and metabolic syndrome 

The nuclear receptor family of PPARs (peroxisome 

proliferator-activated receptor) was named 

after the ability of the original member to induce 

hepatic peroxisome proliferation in mice in response 

to xenobiotic stimuli (14). However, studies 

on the action and structure of the three human 

PPAR isotypes (PPARα, PPARδ, and PPARγ) suggest 

that these moieties are intimately involved 

in nutrient sensing and the regulation of carbohydrate 

and lipid metabolism (14). 

The PPARγ gene produces two proteins, PPARγ1 

and the nearly adipose-specific PPARγ2 (15). 

Expression of PPARγ is highest in adipose tissue, 

where it is the key orchestrator of the transcriptional 

cascade underlying adipocyte differentiation 

(15). PPARγ also plays a key role in the entraining 

of adipose tissue lipid metabolism to nutritional 

state (15). PPARγ is a receptor for the thiazolidinediones 

(TZD), a new class of oral antidiabetics 

(16). It is not yet known whether TZD treatment 

increases subcutaneous fat mass in all body regions 

equally, a question that is of interest in light of the 

burgeoning evidence of important functional metabolic 

differences between upper-body (abdominal) 

and lower-body (including femoro-gluteal) subcutaneous 

fat (17). The PPARγ Pro12Ala rs1801282 

variant is associated with a low body-mass index 

and insulin sensitivity, and it appears to protect 

against the metabolic syndrome. Other relevant 

PPARG (peroxisome proliferator activated receptor 

gamma) gene polimorphisms, are 1431 C>T 

(rs3856806), and recently discovered polymorphisms 

- 689 C>T and -681 C>G (rs10865710) (18). 

The lipin protein family consists of three members, 

lipin-1, lipin-2, and lipin-3. The founding member 

of the family, lipin 1, was identified via positional 

cloning as the mutated gene product in the mouse 

with fatty liver dystrophy (fld). Lipin 1 is expressed 

in a variety of tissues, with the most prominent 

expression in adipose tissue, skeletal muscle, 

and testis (19). Lipin 1 is a multifunctional protein 

that participates in the metabolism of lipids (20, 

21). Lipin 1 acts as a phosphatidate phosphatase 

(PAP) enzyme, which converts phosphatidate 

to diacylglycerol during triglyceride, phosphatidylcholine, 

and phosphatidylethanolamine biosynthesis 

(22, 23). It has been demonstrated that 

lipin 1 can localize to the nucleus of adipocytes 

and hepatocytes, and subcellular localization may 

be influenced by protein phosphorylation (20, 24). 

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78 


Table 1. Primers that were used to analyze parts of PPARG gene with a specific polymorphism 

Name of 

gene 

PPARG 

PPARG 

Name of polymorphism 

1431C> T 

(rs385806) 

Pro12Ala 

(rs1801282) 

The 

length of 

the gene 

259 bp 

247 bp 

The role of nuclear lipin 1 may be related to its 

function as a transcriptional coactivator. Finck et 

al have shown that lipin 1 is required for the activation 

of hepatic fatty acid oxidation genes during 

fasting conditions. Lipin 1 directly interacts with 

the nuclear receptor peroxisome proliferator-activated 

receptor α (PPARα) and PPARγ coactivator 

1α (PGC-1α) in a complex that modulates expression 

of fatty acid oxidation genes (25). The study 

on mice embryonic cells showed that lipin 1α induces 

expression of PPARγ, while lipine 1β induces 

expression of lipogenic genes, such as ACC 

(acyl coenzyme A carboxylase) and diacylglicerol 

acyltransferase (20). Research and studies of this 

gene show an association of its polymorphisms 

with hiperinsulinemia (insulin resistance), while 

the role of the protein in these processes is not yet 

fully understood (26, 27). 

In this study, we hypothesized that genetic variants 

within LPIN1 and PPARG genes were associated 

with traits of metabolic syndrome. This study 

tested a correlation between biochemical parameters 

(including but not limited to, glucose, HbA1c, 

insulin levels, HDL and LDL cholesterol, triglycerides, 

serum proteins, liver enzymes) and frequency 

of polymorphisms in LPIN1 (rs11693809 

and rs2716610) and PPARG gene (rs10865710, 

rs3856806 and rs1801282), in patients with metabolic 

syndrome and type 2 diabetes. 


PPARG and LPIN1 genes polymorphisms have 

been analyzed in patients with metabolic syndrome 

and Type 2 diabetes. 

Patients 

The study included 70 patients of the General Hospital 

Tešanj (2009-2010) diagnosed with metabolic 

syndrome based on the criteria of the International 

Diabetes Federation (IDF) which included measurement 

of waist circumference, blood pressure and 

triglycerides, HDL-cholesterol and serum glucose 

(3). Patients treated with insulin and patients 

with acute infection and / or inflammation were 

excluded from the study. Investigation was done in 

Position of primers Sequence of primer 

PPARG-Ex6_F 

PPARG-ExPPARG-Ex6_R 

PPARG-ExBmism_F 

PPARG-ExBmism_RPPARG-ExBmism_R 

5’-CAGGTTTGCTGAATGTGAAGC-3’ 

5’-TGGCTCAGGACTCTCTGCTAGT-3’ 

5’-CAAGCCCAG TCCTTTCTGTG-3’ 

5’AGCTATGACCAGTGAAGGAATCGCTTTCCG-3’ 

accordance with ethical principles outlined in the 

World Medical Association Declaration of Helsinki 

– Ethical Principles of Medical Research Involving 

Human Subjects (initiated in June 1964, last 

amendment in October 2000). 

Blood samples were collected in the General Hospital 

Tesanj and thereafter stored at -20 C before 

their use. For genetic analysis whole blood was collected 

with EDTA as an anticoagulant. Collected 

blood was kept at -20 C, until a DNA isolation. 

Miller extraction protocol was used for the isolation 

of DNA (37). Total concentration of isolated 

genomic DNA was determined by UV/VIS spectrophotometer 

Nano Drop ND-1000. 

Serum concentrations of insulin and fasting glucose, 

triglycerides, total cholesterol, HDL-cholesterol, 

LDL-cholesterol, albumin, globulin, 

aspartate aminotransferase, alanine aminotransferase, 

γ-glutamyltransferase and bilirubin, creatinine, 

urea and urate, HbA1c and C-reactive 

protein (CRP) were determined. All the analyses 

were performed on VITROS auto analyser 350 

Chemistry System (Ortho-Clinical Diagnostics, 

Heidelberg, Germany). An analysis of IR HOMA 

index was performed on immuno-biochemical 

analyzer Abbott AxSYM (Ilinois, USA). 

Table 2. Composition of the reaction mixture and PCR conditions 

for amplification parts of the PPARG gene containing 

Pro12Ala and 1431C>T polymorphisms 

Polymorphism 

Pro12Ala 1431C>T 

The composition of PCR 

mixture (μl) 

PCR buffer (10x) 2.0 2.0 

dNTP mixture (2 mM) 2.0 2.0 

MgCl2 (25 mM) 2.0 2.4 

F primer (5 μM) 1.0 1.6 

R primer (5 μM) 1.0 1.6 

DNA polymerase (5 U / μl) 0.1 0.1 

DNA (10 ng / μl) 1.0 1.0 

Ultra pure water add 20.0 20.0 

Terms of amplification Temperature / Time 

initial denaturation 95 ° C 10 min. 95 ° C 10 min. 

denaturation 

95 ° C 20 s 95 ° C 20 s 

primer annealing 37 cycles 58 ° C 30 s 57 ° C 30 s 

extension 72 ° C 30 s 72 ° C 30 s 

final extension 72 ° C 7 min 72 ° C 7 min

Table 3. Restriction enzyme used for the analysis of specific 

polymorphisms of PPARG gene 

Polymorphism Restriction enzenzyme 

1431C>T Nla III 

Pro12Ala Hpa II 

Genotyping of Pro12Ala (rs1801282) and 1431C>T 

(rs3856806) polymorphisms in PPARG gene 

A total of two polymorphisms of PPARG gene, 

Pro12Ala (rs1801282) and 1431C>T (rs3856806), 

were analysed by classical PCR. Specific primers 

were used in the process of fragment amplification 

from isolated DNA. Primers that were used 

for amplification, and length of PCR fragments 

are given in Table 1. 

The specificity and quantity of PCR products were 

analysed by electrophoresis on 2% agarose gel. 

PCR product was mixed with loading buffer containing 

xylene-cianol dye and this mix was applied on 

2% agarose gel with 0.01% Sybr Safe. Electrophoresis 

was performed in 1x TAE buffer at 100V for 

20 minutes. Gel was illuminated with UV light and 

photographed with system for documentation and 

analysis of gels G: Box (Syngene). PCR products 

were identified using DNA markers. 

Amplified DNA fragments, or PCR products, 

were exposed to restriction enzymes that have the 

ability to split the PCR products in specific areas. 

In Table 3 the restriction enzymes used for analysis 

Pro12Ala (rs1801282) and 1431C>T (rs3856806) 

polymorphism of PPARG gene are presented. Reaction 

mixture for digestion consisted of: 5 µ l PCR 

product: [Pro12Ala (rs1801282) and 1431C>T 

(rs3856806) polymorphism of PPARG gene], 1x restriction 

buffer, 2 units of restriction enzyme and ultra 

pure water to the total volume of 15 µl. Mixtures 

were incubated in a water bath at 37 °C overnight. 

Restrictions products were analyzed by electrophoresis 

on agarose gel. The entire volume (15 µl) 

of restriction mixture after incubation was mixed 

with 3 µl of loading buffer and applied to 3% 

agarose gel containing 0.01% Sybr Safe. Electrophoresis 

was performed in 1x TAE buffer at 

Figure 1 PCR products of analysis for polymorphism 

1431C>T of gene PPARG 


Table 4. Frequencies in genotypes of analyzed gene polymorphisms 

of genes LPIN 1 and PPARG 

Polymorphisms CC CT TT 

1431 C> T 61 15 3 

SNP 2 32 39 13 

SNP 6 62 22 2 

Polymorphism Pro / Pro Pro / Ala Ala / Ala 

Pro12Ala 55 18 6 

Polymorphism CC CG GG 

-681 C> G 39 28 12 

100V for 40 minutes. Restriction fragments were 

analyzed by the system for documentation and 

analysis of gels G: Box (Syngene). 

Genotyping of -681C>G (rs10865710) of PPARG 

gene polymorphism and SNP2 (rs11693809) and 

SNP6 (rs2716610) of LPIN1 gene 

The polymorphisms -681C>G (rs10865710) 

of PPARG gene, and polymorphisms SNP2 

(rs11693809) and SNP6 (rs2716610) of LPIN1 were 

analyzed by allelic discrimination on the RT PCR 

(Real Time PCR), on cyclic thermostat ABI PRI- 

SM 7700 Sequence Detection System (Applied 

Biosystems). This method also allows quantitative 

analysis of DNA. Method of allele discrimination 

uses two probes, one completely complementary 

to allele without the polymorphism, and another 

complementary to allele with polymorphism. Probes 

were labeled with two different fluorescent 

dyes. In the presence of homozygotes, one probe 

bound only and fluorescence of a single fluorophore 

were detected. In the case of heterozygotes, both 

probes were bound to DNA molecule. In this case, 

the growth of both fluorescences was detected. Primers 

and probes were purchased as the following 

genotyping assays: C__9384417_10 (-681C>G), 

C__2096848_10 (SNP2), C__16280532_10 

(SNP6) (Applied Biosystems). 

Statistical methods 

To compare each biochemical parameter among 

various genotypes of each polymorphism, Kruskal- 

Wallis test was used. For pairwise comparison, 

Mann-Whitney test was used. SPSS 16.0 software 

for Windows was used for statistical analysis. 

Figure 2. PCR products of analysis for polymorphism Pro 12 

Ala of gene PPARG 

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80 


Figure 3. Restriction fragments of 1431C>T polymorphism 

of PPARG gene after exposure of PCR product to restriction 

enzyme NlaIII (3% agarose gel, Syber Safe) (heterozygote CG 

- U51, K36, 10, 65; non-mutated homozygote CC - U50, U55, 

U56, U57, K13, K47, K22, K10, K7, 1; mutated homozygote GG 

– 35; NK - PCR product) 

RESULTS 

Polymorphisms Pro12Ala (rs1801282) and 

1431C>T (rs385806) of PPARG gene were analyzed 

by PCR-RFLP method. Figure 1 shows the 

agarose electrophoresis of amplified PCR fragment 

of 1431 C>T (rs3856806) polymorphism 

in the 6th exon of PPARG gene, which was then 

exposed to restriction NlaIII enzyme (Figure 3). 

Agarose electrophoresis of amplified PCR products 

of Pro12Ala polymorphism in PPARG gene 

is shown in Figure 2, and after restriction with 

HpaII enzyme, in Figure 4. 

Real time PCR method was used for the analysis 

of polymorphisms rs11693809, SNP2 (C>T) and 

rs2716610, SNP6 (C>T) of gene LPIN1, and the 

polymorphism -681 C>T (rs10865710) of PPARG 

gene. Experimental results of these reactions for 

the polymorphism SNP 2 (rs11693809) of gene 

LPIN1 are shown in Figures 5, 6 and 7. The analysis 

of the -681 C>T (rs10865710) polymorphism 

of PPARG gene and the SNP6 (rs2716610) polymorphism 

of LPIN1 gene was carried out in the 

same way as for the case of SNP2 (rs11693809) 

polymorphism of LPIN1 gene. 

Table 4 represents experimental results of all 

analyzed polymorphisms of PPARG and LPIN1 

genes and obtained genotype frequencies. 

Statistical analysis pointed out specific links 

between biochemical parameters and specific genotypes. 

Figure 8 shows statistically significant 

Figure 5. A graphical presentation of the results of real time 

PCR reactions for the CC homozygote of polymorphism SNP2. 

Red curve represents the growth fluorescence of fluorophore 

FAM and green indicates fluorophore VIC ®. The blue curve 

indicates a passive reference ROX. 

Figure 4. Restriction fragments of Pro12 Ala polymorphism of 

PPARG gene after exposure of PCR product to restriction enzyme 

HpaII (3% agarose gel, Syber Safe) (heterozygote CT - 9, 

18, 20, 24; non-mutated homozygote CC – 1, 3, 4, 6, 7, 8, 12, 13, 

25, 27; mutated homozygote GG - 2, 5, 10; NK - PCR product) 

association between insulin values and SNP2 

(rs11693809) polymorphism of LPIN1 gene, 

where patients with CT genotype (heterozygotes) 

had significantly higher values of insulin compared 

to patients with CC genotype (non-mutated 

homozygote) (p G 

(rs10865710) polymorphism of PPARG gene. 

Patients with CG genotype had higher values of 

HDL cholesterol. (P T (rs3856806) polymorphism of PPARG 

gene in patients, where CT genotype had significantly 

higher concentrations of total and LDL 

cholesterol (P < 0.05). 

DISCUSSION 

In a recently published study seven polymorphisms 

in the LPIN1 gene were reported, one of them 

was located in the first intron, being associated 

with plasma insulin values (IVS1 +3341 C>T, 

SNP2, rs11693809) (26). Most of the LPIN1 polymorphisms 

studied so far showed association 

with body mass index (BMI). Greatest association 

with BMI was observed with polymorphism in the 

17th intron (IVS17-228C> T, SNP6, rs2716610) 

(26). For example, rs2716610 was associated with 

BMI in lean Finnish men, and with quantitative 

measures of adiposity in French-Canadian families 

in the Quebec Family Study (26, 28). Another 


PCR reactions for the heterozygote CT of polymorphism SNP2.


PCR reactions for the TT homozygote of polymorphism SNP2 

study in German population (MONICA study Augsburg, 

n = 1,416) found no association between 

rs2716610 and BMI in men or women (29). In our 

study, we have analyzed two of the above mentioned 

LPIN1 polymorphisms. In our study, SNP2 

polymorphism (rs11693809) was associated with 

higher insulin values. Specifically, patients with 

CT genotype had higher levels of insulin compared 

to patients with CC genotype, suggesting an association 

between the mutated T alleles, and higher 

insulin levels. Suviolahti and colleagues reported 

similar results in their study. They found that the 

mutant T allele was associated with higher insulin 

values in male population and higher BMI (26). 

Another study demonstrated the opposite effect of 

polymorphism SNP2 (rs11693809) mutant T allele 

and plasma concentration of SHBG (sex hormone 

binding protein). Patients with this polymorphism 

had lower SHBG, and higher insulin values, which 

is in accordance with inhibition of hepatic SHBG 

synthesis by insulin. (30). No association between 

polymorphism SNP6 (rs2716610) and biochemical 

parametershas were found in this study. However, 

results of a recently published study have shown 

association of mutated T allele and higher triglyceride 

levels, which predicts an increased risk of 

developing insulin resistance (31). 

The presence of mutated G allele of polymorphism 

-681C>G increases the expression of 

PPARγ2, because it lies in the P3 promoter region 

(32). Results of this in vitro study showed 

that presence of mutant G allele of -681C>G polymorphism 

decreased the activation of promotor 

P3 by transcription factor STAT5B, and promo- 

Figure 8. Insulin values in different genotypes of SNP 2 

polymorphism of LPIN 1 gene 

* pG gene PPARG 

* pT (rs3856806) of PPARG gene are associated 

with biochemical parameters of obesity (lower 

body mass index and waist circumference) (35). 

Some studies have linked those polymorphisms 

Figure 10. Concentration of total cholesterol in the different 

genotypes for the polymorphism 1431C>T of gene PPARG 

* p

82 


Figure 11. Concentration of LDL cholesterol in different genotypes 

for the polymorphism 1431C>T of gene PPARG 

* pT 

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CM, Vöhl MC, Nemesh J, Lane CR, Schaffner SF, Bolk 

S, Brewer C, Tuomi T, Gaudet D, Hudson TJ, Daly 

M, Groop L, Lander ES. The common PPARgamma 

between Pro12Ala polymorphism is associated with 

decreased risk of type 2 diabetes. Nat Genet 2000; 

26:76-80. 

Yamauchi T, Kamon J, Waki H, Murakami K, Motojima 

K, Komeda K, Ide T, Kubota N, Terauchi Y, Tobe 

K, Miki H, Tsuchida A, Akanuma Y, Nagai R, Kimura 

S, Kadowaki T. The mechanisms by which both heterozygous 

peroxisome proliferator-activated receptor 

gamma (PPARgamma) deficiency and PPARgamma 

agonist improve insulin resistance. J Biol Chem 2001; 

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Meirhaeghe A, Amouyel P. Impact of genetic variation 

of PPARgamma in humans. Mol Genet Metab 2004; 

83:93-102. 

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of glucose metabolism and reproductive hormones 

in polycystic ovary syndrome on the basis of 

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between Pro12Ala genotype. Hum Reprod 2005; 

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Povezanost polimorfizama PPARG i LPIN1 gena s metaboličkim sindromom i 

dijabetesom tipa 2 

Tamer Bego1 , Tanja Dujic1 , Barbara Mlinar2 , Sabina Semiz1 , Maja Malenica1 , Besim Prnjavorac3,4 , Barbara 

Ostanek2 , Janja Marc2 , Adlija Čaušević1 1 2 Katedra za biohemiju i kliničke analize, Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo, Bosna i Hercegovina; Katedra za 

kliničku biohemiju, Farmaceutski fakultet, Univerzitet u Ljubljani, Ljubljana, Slovenija; 3Opšta bolnica Tešanj, Tešanj; 4Katedra za patofiziologiju, 

Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo; Bosna i Hercegovina. 

SAŽETAK 

Cilj Lipin 1 je nedavno otkriven multifunkcionalni protein, koji učestvuje u metabolizmu lipida na razne 

načine dok je PPARγ glavni kordinator transkripcijskih kaskada za diferencijaciju adipocita, a pored 

toga ima važnu ulogu u metabolizmu lipida tako što omogućava vezivanje i skladištenje slobodnih 

masnih kiselina, te oksidaciju lipida. Polimorfizmi PPARG (peroksisomskim proliferatorom aktivirani 

receptor gama) i LPIN1 gena pokazuju asociranost sa dabetesom tipa 2, metaboličkim sindromom i 

abdominalnom pretilošću. Testirali smo povezanost biohemijskih parametara (glukoze, HbA1c, vrijednosti 

inzulina, HDL i LDL holesterola, triglicerida, serumskih proteina, jetrenih enzima itd.) i polimorfizama 

LPIN1 (rs11693809 i rs2716610) i PPARG (rs10865710, rs3856806 i rs1801282) gena. 

Metode U studiju je bilo uključeno 70 pacijenata kojima je dijagnosticiran metabolički sindrom i dijabetes 

tipa 2. Dva polimorfizma LPIN1 gena (rs11693809 i rs2716610) i tri polimorfizma PPARG gena 

(rs10865710, rs385806 i rs1801282) analizirani su metodom PCR u realnom vremenu (RT-PCR), kao i 

upotrebom klasične PCR-RFLP tehnike. 

Rezultati Analiza je pokazala povezanost polimorfizama rs11693809 LPIN1 gena s vrijednostima inzulina. 

Takođe, rezultati su pokazali povezanost polimorfizama rs10865710 PPARG gena i koncentracije 

HDL holesterola, kao i povezanost polimorfizama rs3856806 PPARG gena i koncentracije ukupnog i 

LDL holesterola kod pacijenata s metaboličkim sindromom i dijabetesom tipa 2. 

Zaključak Rezultati su pokazali asocijaciju analiziranih polimorfizama PPARG i LPIN1 gena s vrijednostima 

HDL, LDL, ukupnog holesterola i vrijednostima inzulina, što ukazuje na važnu ulogu ovih 

gena u metabolizmu lipida i patogenezi dijabetesa tipa 2 i metaboličkog sindroma. 

Ključne riječi: metabolički sindrom, LPIN1, PPARG, dijabetes tipa 2. 

Original submission: 22 October 2010; Revised submission: 26 November 2010; Accepted: 27 November 2010. 

83

84 


Analysis of CYP3A4*1B and CYP3A5*3 polymorphisms in population 

of Bosnia and Herzegovina 

Sabina Semiz 1 , Tanja Dujić 1 , Barbara Ostanek 2 , Besim Prnjavorac 1,3 , Tamer Bego 1 , Maja Malenica 1 , 

Barbara Mlinar 2 , Janja Marc 2 , Adlija Čaušević 1 

1Department for Biochemistry and Clinical Analysis, Faculty of Pharmacy, University of Sarajevo, Sarajevo, Bosnia and Herzegovina, 

2 3 Department for Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Slovenia, General Hospital Tesanj, Tesanj, Bosnia 

and Herzegovina. 


Sabina Semiz 

Department for Biochemistry and Clinical 

Analysis 

Faculty of Pharmacy, University of 

Sarajevo 

Koševska 4 (Čekaluša 90), 

71000 Sarajevo, Bosnia and Herzegovina 

Phone/fax.: +387 33 269 640 

E-mail: sabinasemiz@hotmail.com 




07 December 2010; 

Accepted: 

08 December 2010. 

ABSTRACT 


Aim Differences in the frequency of distribution of the cytochrome 

P450 (CYP) allelic variants have been demonstrated between 

distinct ethnic groups, contributing to observed interindividual 

variation in drug response. In this study we determined, 

for the first time, prevalence of the common allelic variants of the 

polymorphic CYP enzymes, CYP3A4*1B and CYP3A5*3, in the 

population of Bosnia and Herzegovina (BH). 

Methods Genomic DNA was extracted from blood samples collected 

from 140 unrelated subjects. A real-time PCR was used for 

the detection of CYP polymorphisms, with the application of the 

specific TaqMan® SNP Genotyping Assay (Applied Biosystems) 

for CYP3A5*3, while CYP3A4*1B was genotyped by high-resolution 

melting analysis. 

Results Our results have shown that the distribution of CYP3A4*1B 

and CYP3A5*3 alleles was in line with the data reported in European 

Caucasians. We confirmed that CYP3A4*1B mutant allele is 

rare in Caucasians, being present in only 5.1% individuals. However, 

CYP3A5*3 polymorphism was found to be predominant in the 

Bosnian population with an incidence of 94%, similarly to other 

European populations tested so far. Interestingly, we have demonstrated 

a strong linkage disequilibrium between CYP3A5*3 and 

CYP3A4*1B alleles. No significant difference in allele frequencies 

for CYP3A4*1B and CYP3A5*3 has been shown between male 

and female subjects participating in our study. 

Conclusion Our data demonstrated the high prevalence of 

CYP3A5*3 allele in Bosnian population, indicating significance 

of analysis of CYP3A5 and CYP3A4 polymorphisms and corresponding 

allele frequencies in specific ethnic groups. Importantly, 

results of this study may lead to translation of pharmacogenetics 

and individualized therapeutic approach in current clinical practices 

in BH. 

Key words: Cytochrome P450, CYP3A4, CYP3A5, Bosnian, 

pharmacogenetics.

INTRODUCTION 

Drug-metabolizing enzymes (DME), which 

include phase I and II metabolizing enzymes, 

play a central role in the intestinal absorption/ 

permeability, metabolism, elimination, and detoxification 

of various drugs. Cytochrome P450 

(CYP) enzymes are major phase I metabolizing 

enzymes, which play particularly important role 

in disposition of various drugs (1). The human 

CYP superfamily contains 57 functional genes 

and 58 pseudogenes, where CYP2C8, CYP2C9, 

CYP2C19, CYP2D6, CYP3A4, CYP3A5, and 

CYP1A2 metabolize around 90% of drugs (2,3). 

CYP3A4 and CYP3A5 are considered as major 

functional CYP3A isoforms in human liver and 

intestine (4). They regulate metabolism of approximately 

50% of commonly used drugs, such 

as cholesterol-lowering drugs, calcium channel 

antagonists, immunosuppressants, antibiotics, 

oral anticoagulants, and anticancer chemotherapeutic 

drugs (5). 

Importantly, CYP-regulated drug metabolism is 

prone to genetic variability that can result in an 

enzyme with normal, low, or no activity. CYP 

polymorphisms are responsible for observed variations 

in drug response among patients with different 

ethnic origins (5,6), which could be combined 

with genetic variations in other DME, drug 

transporters, and drug receptors. Interindividual 

variation in CYP3A expression is substantial and 

earlier studies suggest that 30-90% of these differences 

in hepatic CYP3A activity is due to genetic 

factors, such as single nucleotide polymorphisms 

(SNPs) (7,8). Around eighty SNPs of CYP3A4/5 

have been reported to the Human P450 Allele 

Nomenclature Committee (www.imm.ki.se/CY- 

Palleles). CYP3A protein expression in liver and 

small intestine varies up to 40-fold, leading to 

variation in drug metabolism (9,10). 

Thus, a number of CYP3A4/5 polymorphisms 

have been described, with a few of them occurring 

frequently enough to contribute to variations 

in CYP3A activity. For instance, the CYP3A4*1B 

is a common SNP located in the 5′ promoter region, 

which probably has an effect on transcription, 

so interindividual variability in CYP3A4 activity 

may be the result of transcriptional regulation 

(11-14). Functional SNPs are more commonly 

observed in the CYP3A5 gene. The CYP3A5*3 

SNP in intron 3 causes alternative splicing and 

protein truncation (15). This mutant allele is considered 

as the major defective CYP3A5 allele and 

may play an important role in interindividual and 

interethnic differences in the metabolic profile 

of many drugs (4,16). Location and effects of 

CYP3A4*1B and CYP3A5*3 polymorphisms are 

summarized in Table 1. Interestingly, previous 

studies have shown significant linkage disequilibrium 

between CYP3A5*3 and CYP3A4*1A 

in Caucasians, and between CYP3A5*1 and 

CYP3A4*1B in African-Americans (17,18,20). 

The distribution of the common variant alleles of 

CYP genes, such as CYP3A4*1B and CYP3A5*3, 

varies among different ethnic populations. Particularly, 

CYP3A5*3C frequency shows dramatic 

interethnic variation. In Europeans, the 

CYP3A5*3C variant seems to be the predominant 

allele (around 94% frequency), but this allele has 

a much lower frequency in the African and African 

American population (12-36%) (5,20). The 

allelic frequency of CYP3A4*1B in Caucasians 

and African Americans is around 4-9% and 59- 

79%, respectively (5,20,21). Interestingly, previous 

studies also suggest gender differences in 

CYP3A expression and activity (20, 22). 

Since the frequency of both CYP3A5*3 and 

CYP3A4*1B allele have not been determined 

yet in the population of Bosnia and Herzegovina 

(BH) and has a potential impact in the pharmacogenetic 

applications, in this study we have determined 

the frequency of these mutant alleles in a 

representative group of 140 BH individuals. 


Subjects 

In this study we have analyzed specific CYP polymorphisms 

in a group of 140 unrelated individuals 

(95 females and 45 males), which were 

patients of the General Hospital in Tešanj, BH. 

Polymorphism 

Semiz et al CYP3A4/5 variations in BH 

Table 1. Locations and effects of CYP3A4*1B and CYP3A5*3 

polymorphisms 

Nucleotide 

change 

rs number* 

Location, 

protein 

effect 

CYP3A4*1B -392A>G rs2740574 Promoter 

CYP3A5*3 6986A>G rs776746 

Splicing 

defect 

Functional 

effect 

Probable 

effect on 

transcription 

Decreased 

expression 

and activity 

*rs number, reference Single Nucleotide Polymorphism (SNP) ID 

assigned by the SNP database at National Center for Biotechnology 

Information (dbSNP) 

85

86 


The mean age of the subjects was 54±1.2 years 

(range of 30-70 years old). Blood samples were 

obtained from all participants in fasting conditions 

from antecubital vein into siliconized tubes 

(BD Vacutainer Systems, Plymouth, UK). 

All research involving human subjects and material 

derived from human subjects in this study 

was done in accordance with the ethical recommendations 

and practices of the General Hospital 

in Tešanj and complied with ethical principles outlined 

in the World Medical Association Declaration 

of Helsinki – Ethical Principles for Medical 

Research Involving Human Subjects (initiated in 

June 1964, last amendment in October 2000). 

Genotyping Analysis 

Genomic DNA was extracted from blood samples 

by Miller’s salting-out procedure (23). The 

total concentration of isolated genomic DNA was 

determined by UV/VIS spectrofotometer Nano- 

Drop ND-1000. Polymorphism CYP3A5*3 was 

genotyped by hydrolysis probes using TaqMan® 

SNP Genotyping Assay (Applied Biosystems), 

assay ID C__26201809_30. Real-Time PCR was 

performed on the ABI PRISM 7000 Sequence 

Detection System (Applied Biosystems). Polymorphism 

CYP3A4*1B was genotyped by the 

optimized high-resolution melting analysis on 

the LightCycler ® 480 Real-Time PCR System 

(Roche Diagnostics). 

Statistical Analysis 

Statistical data analysis was done using SPSS 

Statistics 17.0. Statistical significance was set 

as p0,05). 

As shown in Table 2, the homozygotes for the A 

allele for CYP3A4*1B were predominantly present 

(94.9%), while the homozygotes for the G 

allele were not detected in our study group. The 

frequency distribution for CYP3A5*3 showed 

that homozygotes for the G allele were predominant 

(86,3%), and homozygotes for the A allele 

were not found in the same group of participants. 

All seven subjects which were heterozygous 

for CYP3A4*1B were also heterozygous for 

CYP3A5*3 allele (Table 3). Thus, CYP3A4*1B 

and CYP3A5*3 polymorphisms were in 100% linkage 

disequilibrium (D’=1.0) in our study group. 

We also analysed a possible gender differences 

in the prevalence of CYP3A4*1B and CYP3A5*3 

polymorphisms. However, no significant difference 

in allele frequencies for CYP3A4*1B and 

CYP3A5*3 polymorphisms was demonstrated 

between male and female subjects participating 

in our study (data not shown). 

DISCUSSION 

Despite the marked advances in drug therapy, significant 

fractions of patients do not respond favorably 

or experience severe adverse drug effects. 

Table 3. Simultaneous existence of different genotypes of 

CYP3A4*1B and CYP3A5*3 polymorphisms detected in the 

same subjects from BH 

CYP3A4*1B/ CYP3A5*3 Number of subjects (%) 

AA/GG 117 (86,0%) 

AA/AG 12 (8,8%) 

AG/AG 7 (5,1%)

Pharmacogenetic studies have shown that polymorphisms 

of drug-metabolizing enzymes, transporters 

and receptors contribute to variable drug 

response (1). CYP3A4 and CYP3A5 isoenzymes 

are responsible for the metabolism of over 50% 

of all clinically used drugs, including commonly 

used antidepressants, antibiotics, antihipertensives, 

steroids, and immunosuppressants (5). Thus, 

polymorphisms in the CYP family may have the 

most impact on the fate of these and other therapeutic 

drugs whose metabolism they regulate. 

The frequency of CYP3A variants, including 

CYP3A4*1B and CYP3A5*3 polymorphisms, 

varies substantially in different populations 

(5,20,21). Our results have demonstrated that 

the prevalence of these defective alleles in population 

of Bosnia and Herzegovina were in line 

with the data reported earlier for other European 

populations of Caucasian origin. Here we confirmed 

that CYP3A4*1B mutant allele is rare in 

Caucasian population, with AG genotype being 

present in only 5.1%, while GG genotype was not 

detected in the Bosnian population group. Similarly, 

previous studies have detected CYP3A4*1B 

allele in about 4-9% Caucasians (20,21), being 

much lower than in African-Americans in which 

the frequency of this allele was reported to be 

around 53% (4,20-22,25). 

In contrast, CYP3A5*3 polymorphism was found 

to be predominant in the Bosnian population with 

an incidence of 94.9%. This allele frequency is 

in accordance with previous studies performed 

in other European populations, such as 94.35% 

in Greek (5), 94% in British (16), and 91.7% in 

Dutch (26), while to some extent different from 

the Portuguese (87.5%) (21) and French study 

group (82%) (27). 

Furthermore, here we have demonstrated for the 

first time a simultaneous existence of specific genotypes 

of CYP3A4*1B and CYP3A5*3 polymorphisms 

in the same individuals participating in 

our study. All subjects with mutant CYP3A4*1B 

allele were also defective for CYP3A5*3 allele. 

Thus, our data complement data from previous 

studies, which indicated a strong linkage disequilibrium 

between CYP3A5*3 and other mutant 

CYP3A4 alleles (CYP3A4*1A) in Caucasians 

(17,18), while a recent meta-analysis demonstrated 

a linkage disequilibrium between CYP3A5*1 

and CYP3A4*1B in African-Americans (20). 


The clinical significance of CYP3A4*1B and 

CYP3A5*3 polymorphisms is still under investigation. 

The variant frequency of this polymorphism 

among different ethnic groups may contribute 

significantly to drug efficacy and toxicity. 

Recently, CYP3A5*3 polymorphism has been 

suggested to be implicated in variable response 

to treatment with statins (HMG-CoA reductase 

inhibitors) (28), which are today one of the most 

prescribed drugs globally. Furthermore, it has 

been reported that the bioavailability of immunosuppressant 

drug cyclosporine, which is metabolized 

by CYP3A4 and CYP3A5, was significantly 

lower in African-American than in Caucasian- 

American patients, with a preceding group of 

patients requiring higher cyclosporine dose (29). 

This difference in the cyclosporine dosing may 

be due to reported ethnic difference in CYP3A5*3 

allele frequency, which was found to be significantly 

lower in African-American (47.5%) than 

in Caucasian population (91.7%) (29). In addition, 

the use of the CYP3A5 genotype to predict 

the optimal dose of another immunosuppressive 

drug, tacrolimus, is also highly recommended by 

recent studies (30,31). The effects of CYP3A5*3 

genotype on metabolism and disposition of commonly 

used calcium channel blockers, such as 

nimodipine (32) and amlodipine (33), were also 

recently reported. Remarkably, polymorphisms 

in CYP3A genes, such as CYP3A5 and CYP3A4, 

have also been associated with several disease 

conditions, including prostate, lung, and breast 

cancer, secondary leukemia, diabetes, and hypercholesterolemia 

(17,19,34). 

In conclusion, our data demonstrated the high 

prevalence of CYP3A5*3 allele in Bosnian population, 

similarly to frequency of this variant allele 

found in other studies performed in European 

Caucasians. Here we have also showed the simultaneous 

existence of heterozygous CYP3A4*1B 

and CYP3A5*3 in the same group of subjects. 

Since CYP3A4 and CYP3A5 are involved in 

disposition of many currently often used drugs, 

it is pertinent to analyse CYP3A4 and CYP3A5 

polymorphisms and determine corresponding allele 

frequencies in specific ethnic groups. In line 

with this, the results of our study may represent 

the basis for the development of a pharmacogenetic 

program and guide national planning for the 

selection of therapeutic options in BH. 

87

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ACKNOWLEDGEMENTS/ DISCLOSURES 

Authors thank all the subjects who participated in 

the study, medical doctors, and paramedical staff 

from the General Hospital Tešanj who assisted in 

the study. 

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1. Eichelbaum M, Ingelman-Sundberg M, Evans WE. 

Pharmacogenomics and individualized drug therapy. 

Annu Rev Med. 2006; 57:119-37. 

2. Wilkinson GR. Drug metabolism and variability 

among patients in drug response. N Engl J Med 

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104. 

Analiza CYP3A4*1B i CYP3A5*3 polimorfizama u populaciji iz Bosne i Hercegovine 

Sabina Semiz1 , Tanja Dujić1 , Barbara Ostanek2 , Besim Prnjavorac1,3 , Tamer Bego1 , Maja Malenica1 , 

Barbara Mlinar2 , Janja Marc2 i Adlija Čaušević1 1 2 Katedra za biohemiju i kliničke analize, Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo, Bosna i Hercegovina; Katedra za 

kliničku biohemiju, Farmaceutski fakultet, Univerzitet u Ljubljani, Ljubljana, Slovenija; 3Opšta bolnica Tešanj, Tešanj, Bosna i Hercegovina 

SAŽETAK 

Cilj Postojanje značajnih razlika u frekvenciji različitih alelskih varijanti citohroma P450 (CYP) među 

različitim etničkim grupama doprinosi interindividualnoj razlici odgovora na terapiju lijekovima. Ovo je 

prva studija u kojoj smo analizirali prevalencu alelskih varijanti polimorfnih CYP enzima, CYP3A4*1B 

i CYP3A5*3, u populaciji iz Bosne i Hercegovine (BiH). 

Metode Genomska DNK izolovana je iz uzoraka krvi, uzetih od 140 nesrodnih osoba. U cilju detekcije 

navedenih CYP polimorfizama korištena je metoda RT-PCR, uz aplikaciju specifičnih TaqMan® SNP 

testova (Applied Biosystems) za CYP3A5*3, dok je CYP3A4*1B genotipiziran uz korištenje analize 

taljenja DNK visoke rezolucije. 

Rezultati Rezultati ove studije pokazali su da je distribucija CYP3A4*1B i CYP3A5*3 alela u skladu s 

podacima ranije objavljenim za evropsku populaciju bjelaca. Potvrdili smo da je CYP3A4*1B mutant 

alela rijetka kod bjelaca i prisutna kod samo 5.1% individua. Međutim, naši su rezultati pokazali da je 

CYP3A5*3 polimorfizam dominantan kod bosanskohercegovačke populacije, s incidencom od 94%, što 

je slično nalazima u drugim testiranim evropskim populacionim grupama. Interesantno je da smo zabilježili 

značajnu neravnotežu veze (linkage disequilibrium) između CYP3A5*3 i CYP3A4*1B alela. Nije 

primjećena signifikantna razlika u frekvenciji CYP3A4*1B i CYP3A5*3 alela između osoba muškog i 

ženskog spola koje su učestvovale u našoj studiji. 

Zaključak Naši rezultati pokazali su visoku prevalencu CYP3A5*3 alele u bosanskohercegovačkoj populaciji, 

demonstrirajući značaj analize CYP3A4 i CYP3A5 polimorfizama i frekvencije odgovarajućih 

alela kod specifičnih etničkih grupa. Važno je istaći da rezultati ove i sličnih budućih studija, mogu dovesti 

do bržeg uvođenja farmakogenetike i individualiziranog terapeutskog pristupa u sadašnju kliničku 

praksu u BiH. 

Ključne riječi: Citohrom P450, CYP3A4, CYP3A5, BiH, farmakogenetika. 


89

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Optimisation of methods for quantifying plasma mRNA levels 

from genes responsible for coronary artery plaque development 

and destabilization 

Darko Černe 1 , Irma Štern 1 , Igor Kranjec 2 , Janja Marc 1 

1 Faculty of Pharmacy, University of Ljubljana, 2 Department of Cardiology, University Medical Centre Ljubljana; Ljubljana, Slovenia 


Darko Černe 

Faculty of Pharmacy, 

University of Ljubljana 

Aškerčeva 7, 1000 Ljubljana, Slovenia 

Phone: +386 1 47 69 644; 

Fax: +386 1 42 58 03 

E-mail: darko.cerne@ffa.uni-lj.si 





Accepted: 

05. December 2010. 

ABSTRACT 


Aim To investigate the hypothesis that in patients with coronary 

atherosclerosis it is possible to measure plasma mRNA levels from 

genes responsible for plaque development and destabilization. 

Methods Methods for RNA isolation, mRNA transcription and 

quantitative PCR were evaluated and optimised, in order to achieve 

reliable mRNA quantification. 

Results mRNA level was possible to quantify from plasma of 

patients with coronary atherosclerosis, as well as from healthy 

volunteers, from genes encoding cathepsin S, cathepsin B, CD40 

molecule, monocyte chemotactic protein 1, death-associated protein 

kinase 1, matrix metallopeptidase 9, vascular cell adhesion 

molecule 1 and phosphoglycerate kinase 1 (reference gene). Analytical 

between-run imprecision of average threshold cycle, expressed 

as coefficient of variation was below 2%. EDTA blood samples 

should be centrifuged within one hour of venesection. It was 

not possible to quantify plasma mRNA level from genes encoding 

macrophage scavenger receptor 1, perilipin, tissue factor, phospholipase 

A2 group IIA, collagen type I alpha 2 and interleukin 1 

alpha. 

Conclusion Further plasma mRNA analysis is reasonable to access 

its potential usefulness in non-invasive in vivo monitoring of 

gene expression profile in vascular beds. 

Key words: plasma mRNA, coronary atherosclerosis, method optimisation

INTRODUCTION 

Cell-free nucleic acids (DNA, RNA) can be found 

in plasma, serum and cell-free fractions of 

various other biological fluids, such as urine and 

bronchial lavage. Although discovered in the 

circulation in 1948 (1), the finding remained almost 

unrecognized until 1966, when high levels 

of DNA were found in plasma of patients with 

systemic lupus erythematosus (2). Nowadays, 

quantitative and qualitative analysis of cell-free 

nucleic acids obtained from body fluids is considered 

to be clinically useful in non-invasive prenatal 

diagnosis (3), and in diagnosis and monitoring 

of numerous cancers (4). Novel applications 

are emerging also in other critical care settings, 

such as myocardial infarction (5, 6). 

Contemporary research in atherogenomics is focused 

on identifying genes typically expressed in 

individual atherosclerotic plaque cells, at different 

stages of atherogenesis or in different pathomorphological 

forms of the disease. For instance, 

turbulent, as opposed to laminar blood flow damages 

endothelial cells and causes over-expression 

of many genes, such as vascular cell adhesion 

molecule 1 (VCAM1), E-selectin, monocyte 

chemotactic protein-1 (CCL2) and thrombospondin-1 

(7). By comparing unstable (ulcerated surface 

with or without thrombosis or hemorrhage) 

and stable (smooth luminal surface indicating 

intact fibrous cap) segments of individual plaques, 

several genes expected to be associated with 

unstable plaque were found to be over-expressed, 

including matrix metallopeptidase 9 (MMP9) and 

cathepsin S and B (CTSS, CTSB) (8). Analysis 

of gene expression profiles in coronary plaques 

from patients with unstable angina showed higher 

tissue factor (F3), but lower anticoagulant 

protein S, cyclooxygenase, interleukin-7, CCL2 

and CCL8 expression than from patients with stable 

angina (9). In vascular smooth muscle cells of 

stent stenosis several genes were over-expressed, 

including collagen type I alpha 2 (COL1A2), cathepsin 

D and heat shock protein 27 (10). 

The existence of cell-free mRNA in plasma of 

patients with atherosclerosis has not been reported. 

It was hypothesised that, in patients with coronary 

atherosclerosis, it is possible to measure 

plasma mRNA level from genes encoding CTSS, 

CTSB, CD40 molecule (CD40), CCL2, deathassociated 

protein kinase 1 (DAPK1), MMP9, 

Černe et al Plasma mRNA in coronary atherosclerosis 

VCAM1, macrophage scavenger receptor 1 

(MSR1), perilipin (PLIN), F3, phospholipase A2 

group IIA (PLA2G2A), COL1A2 and interleukin 

1 alpha (IL1A), i.e. from genes responsible for 

plaque development and destabilization. If the 

answers are positive, it would mean that plasma 

mRNA estimation might offer a non-invasive in 

vivo assessment and monitoring of gene expression 

profile in vascular beds. 

MATERIALS AND METHODS 

Subjects and blood sampling 

Patients with stable or unstable angina (SA or 

UA) or acute myocardial infarction (AMI) were 

selected at random from patients hospitalized at 

the Department of Cardiology in the Medical 

Centre Ljubljana (n = 38, median age 62 years, 

69 % males). All patients had angiographically 

proven coronary atherosclerosis. Statin treatment 

(no/yes) was recorded. Smokers, hypertensive 

patients and patients with diabetes were not excluded 

from the study. Healthy subjects (n = 23, 

median age 52 years, 63 % males) were selected 

at random from volunteers having no subjective 

problems and being at less than 10 % risk of an 

ischemic coronary event in the following 10 years 

(11). The study was approved by the national 

ethics committee. Informed consent was obtained 

from all participating subjects. 

Peripheral blood was taken into EDTA tubes 

(Becton Dickinson, Plymouth, UK) after at least 

12 hours of fasting. To reduce the bias resulting 

from instability of mRNA the same double-spin 

procedure was used in all subjects to obtain plasma 

nucleic acids. Sample processing took place 

within 1 h of collection. EDTA blood was first 

centrifuged at 1600 x g at 4 ˚C for 10 min. Plasma 

was carefully transferred into a new tube, 

followed by a second centrifugation at 16000 x g 

at 4 ˚C for 10 min. In both pipettings plasma 0.5 

cm above the buffy coat or above the bottom of 

the tube was further processed. Haemolysed samples 

were not used for analysis. Cell-free plasma 

was stored in liquid nitrogen up to four weeks for 

RNA isolation. 

RNA isolation and mRNA transcription to cDNA 

RNA was isolated from 200 μL of cell-free plasma 

using QIAamp MinElute Virus Spin Kit (Qiagen, 

91

92 


Hilden, Germany; purchased in 2008), according 

to the manufacturer’s recommendations, but with 

certain modifications: 1) the incubation time for 

proteolysis was 5 min; 2) RNA was eluted from the 

column with 55 RNase-free water and the collected 

eluate used for the second elution of RNA from the 

same column. If not used immediately for further 

processing, RNA eluate was stored at -80 ˚C. 

Eluted mRNA was transcribed to cDNA with SuperScript 

VILO cDNA Synthesis Kit (Invitrogen, 

Carlsbad, CA, USA; purchased in 2008). First, 

RNA eluate was incubated in a water bath (65 

˚C) for 5 min, then immediately placed on ice for 

another 5 min. The manufacturer’s protocol was 

followed from then on. cDNA solution was stored 

at -80 ˚C until processed. 

cDNA quantification 

The following TaqMan Gene Expression Assays 

(Applied Biosystems, Foster City, CA, 

USA) were used for cDNA transcript quantification: 

Hs00175403_m1 for CTSS (exon boundary 

3-4, amplicon lengh 82), Hs00947433_m1 

for CTSB (exon boundary 2-3, amplicon lengh 

73), Hs00374176_m1 for CD40 (exon boundary 


for CCL2 (exon boundary 1-2, amplicon lengh 

101), Hs00234489_m1 for DAPK1 (exon boundary 


for MMP9 (exon boundary 1–2, amplicon lengh 

79), Hs01003369_m1 for VCAM1 (exon boundary 


for MSR1 (exon boundary 9-10, amplicon lengh 

67), Hs00160173_m1 for PLIN (exon boundary 

2-3, amplicon lengh 54), Hs00175225_m1 for 

F3 (exon boundary 3-4, amplicon lengh 118), 

Hs00179898_m1 for PLA2G2A (exon boundary 

5-6, amplicon lengh 100), Hs00164099_m1 for 

COL1A2 (exon boundary 6-7, amplicon lengh 68) 

and Hs00899848_m1 for IL1A (exon boundary 5-6, 

amplicon lengh 128). Phosphoglycerate kinase 1 

(PGK1; Hs99999906_m1; exon boundary 4-5, amplicon 

lengh 75; Applied Biosystems) was used as 

a reference gene. cDNAs were amplified by PCR 

according to the manufacturer’s protocol (Roche 

Diagnostics, GmbH, Mannheim, Germany), using 

volumes from 1.5 μL to 3.75 μL and the kit components 

in a final reaction volume of 25 μL. 

For every commercially available TaqMan Gene 

Expression Assay lot number (Applied Biosy- 

stems) the possibility of DNA interference was 

excluded by running PCR reaction with subjects’ 

samples but omitting the reverse transcription. 

Alternatively, DNA interference could be excluded 

by an analysis of the size of the amplified 

PCR products on agarose gel electrophoresis or 

by running a PCR reaction with human DNA. All 

reactions were run in triplicates and the average 

threshold cycle of triplicates (Ct) calculated. To 

exclude the possibility of external mRNA contamination, 

a negative control (blank) was analysed 

in each PCR run. The between-run imprecision 

for the whole analytical procedure (from RNA 

isolation to transcript quantification), expressed 

as the coefficient of variation (KV) of Ct, was 

less than 2 %. For clinical evaluation, the amount 

of each investigated transcript was standardized 

to the amount of a reference gene (12). 


When compulsory, the differences between experiments 

were tested with Wilcoxon matched-pair 

test. A statistical analysis was carried out with 

SPSS v.17.0 (SPSS Inc., Chicago, Illinois, USA). 

Excel 2007 was used for figure constructions 

(Microsoft Corporation, Redmond, Washington, 

USA). 

RESULTS 

Four different methods for RNA isolation from 

plasma were compared: QIAamp UltraSens Virus 

Kit (Qiagen), EZ1 Virus Mini Kit v2.0 (Qiagen), 

TRIzol method (13) followed by the use of 

RNeasy Plus Mini Kit (Qiagen) and QIAamp MinElute 

Virus Spin Kit (Qiagen). The best results 

were obtained with QIAamp MinElute Virus Spin 

Kit, based on the yield of extracted RNA from 

plasma and the within-run precision in quantitative 

RT-PCR followed the RNA extraction. Regardless 

of the method used, RNA 6000 Nano assay 

kit (Agilent Technologies, Walbdronn, Germany) 

was ineffective in assessing quality and quantity 

of isolated RNA. 

The manufacturer’s protocol for QIAamp MinElute 

Virus Spin Kit was additionally modified 

to improve the yield of extracted RNA and the 

detection rate and within-run precision in the subsequent 

quantitative RT-PCR. To summarize the 

most important experiments: 1) second elution 

of the column, with the eluate from the first elu-

tion of the column with 55 μL of RNase-free water, 

yielded approximately two Ct lower values 

in the following quantitative RT-PCR than single 

elution with 55 μL of RNase-free water; 2) after 

a single elution with 30 μL of RNase-free water, 

approximately half the RNA remained on the column; 

3) after applying the eluting solution to the 

column, 5 min incubation period before centrifugation 

is strongly advisable, as is also suggested 

by the manufacturer; and 4) 5 min proteolysis 

gave the lowest Ct values and the better detection 

rate and within-run precision in the following quantitative 

RT-PCR than 15 or 25 min proteolysis. 

Furthermore, the optimal time for proteolysis 

appears to depend on the clinical status of the subject 

(healthy subject, patient with SA or UA or 

AMI) and on the investigated transcript, but in 

general, 5 min proteolysis was superior. 

The protocol for mRNA transcription to cDNA, 

as described in Materials and methods, offered 

better quantitative PCR performance than the 

protocol of High Capacity cDNA Archive Kit 

(Applied Biosystems), which was also extensively 

studied in our experiments. Pre-treatment 

of RNAs at 65 ˚C for 5 min also fundamentally 

improved the performance of the subsequent quantitative 

PCR reaction (data not shown). 

For each transcript, if inventoried reagents were 

available (Applied Biosystems), amplicons near 

the 5’-end, 3’-end and in the middle of the transcript 

were tested for the most reliable mRNA 

quantification. In general, the most satisfactory 

was the use of 5’-end amplicons, which yielded 

the best detection rate, the highest plasma mRNA 

concentration and the best within-run precision. 

In contrast, the worst detection rate and withinrun 

precision were obtained with use of the 3’end 

amplicons. 

Eleven reference genes were tested using the Taq- 

Man Human Endogenous Control Plate (Applied 

Biosystems), according to the manufacturer’s 

protocol and by careful selection of samples to 

ensure constant expression of the selected gene 

regardless of experimental conditions, including 

variations in stage of disease, patient treatment 

and presence of risk factors (diabetes, smoking, 

male gender). The reference gene was selected 

on the basis of the smallest geometric average 

threshold Ct of individual candidates and results 

from geNorm (14). The most appropriate referen- 


ce genes were PGK1 and cyclophilin A. PGK1 

was also an appropriate reference gene in patients 

undergoing percutaneous coronary revascularization. 

With the use of fully optimised analytical procedure 

(Table 1) it has been found that in patients 

with coronary atherosclerosis, as well as in healthy 

subjects, it is possible to quantify plasma 

mRNA level from genes encoding CTSS, CTSB, 

CD40, CCL2, DAPK1, MMP9, VCAM1 and 

PGK1 with analytical between-run imprecision 

less than 2%. For example, in an SA patient 

without statin treatment, Ct values of three independent 

mRNA quantifications (from RNA isolation 

to transcript PCR quantification) on three 

consecutive days were: for CATS 34.34, 33.08 

Table 1. Recommendations for plasma mRNA quantification 

from genes responsible for coronary artery plaque development 

and destabilization 

Protocol step Reagents, sample 

Comments, recommendations 

Processing EDTA blood Store at 4 °C 

after 

venesection Centrifuge within one hour 

(not within few minutes) 

Check clinical status specific 

influences on mRNA stability 

mRNA QIAamp MinElute Use 5 min proteolysis 

isolation Virus Spin generally 

from plasma Kit (Qiagen) Elute column with 55 μL of 

RNase-free water 

Re-elute the column with the 

first eluate 

Use 5 min incubation period 

after applying the eluting 

solution to the column 


influences on the optimal 

proteolysis time 

mRNA SuperScript VILO Heat mRNAs to 65 ˚C for 5 

transcription cDNA Synthesis min and place immediately 

Kit (Invitrogen) on ice for another 5 min before 

running the transcription 

protocol 

mRNA TaqMan Gene Use of 5’-end amplicons of 

quantification Expression Assays 

and TaqMan 

quantitative 

the transcript generally 

PCR (Applied For every TaqMan Gene 

Biosystems) Expression Assays lot number 

confirm that DNA does 

not interfere 


influences on the optimal 

amplicon target site selection 

Reference Phosphoglycerate Appropriate for between 

gene kinase 1 or cyc- subject comparisons (corolophilin 

A (Applinary artery disease patients, 

ed Biosystems) healthy subjects) 

Appropriate for following 

patients undergoing percutaneous 

coronary revascularization 

93

94 


and 34.14 (KV 0.40 %), for CCL2 38.27, 37.89 

and 38.12 (KV 0.50 %), for DAPK1 35.94, 36.17 

and 35.83 (KV 0.48 %) and for PGK1 32.94, 

33.17 and 32.93 (KV 0.41 %). Therefore, plasma 

mRNA levels could be monitored continuously 

in patients undergoing percutaneous coronary 

revascularisation (Figure 1). On the contrary, 

plasma mRNA level from genes encoding MSR1, 

PLIN, F3, PLA2G2A, COL1A2 and IL1A could 

not be quantified. 

Plasma mRNA levels in EDTA whole blood stored 

at 4 °C decreased rapidly, regardless of the 

measured transcript and amplicon target site selection 

(Figure 2). The decay rate was different 

in different mRNA species and dependent on a 

clinical status of the subject. 

DISCUSSION 

Our study has shown that in patients with coronary 

atherosclerosis, as well as in healthy subjects, 

it is possible to quantify plasma mRNA 

level from genes encoding CTSS, CTSB, CD40, 

CCL2, DAPK1, MMP9, VCAM1 and PGK1 with 

analytical between-run imprecision less than 2 

%. With the use of current molecular methods 

and techniques we were unable to quantify plasma 

mRNA level from genes encoding MSR1, 

PLIN, F3, PLA2G2A, COL1A2 and IL1A. 

Plasma RNA isolation with QIAamp MinElute 

Virus Spin Kit proved a reliable background for 

mRNA quantification, but the manufacture’s protocol 

can be modified easily to further improve 

the performance. It functions better than the TRIzol 

method, followed by the RNeasy Plus Mini 

Kit, mostly used nowadays (13, 15-18), and also 

Figure 1. Time dependent contour of plasma CCL2 mRNA 

level (standardised to plasma PGK1 mRNA level) in patient 

undergoing percutaneous coronary revascularisation* 

*CCL2, gene encoding monocyte chemotactic protein 1; PGK1, 

gene encoding phosphoglycerate kinase 1. 

better than QIAamp UltraSens Virus Kit, which 

we optimised in our previous research (19). The 

improved performance with 5 min over 15 or 25 

min proteolysis cannot be explained rationally. 

Plasma mRNA is mostly particle associated, with 

various plasma proteins (15) and in apoptotic bodies 

(20). It is reasonable to believe that in our 

protocol with short proteolysis we measure predominantly 

mRNA associated with minor protein 

complexes. In this respect, it is notable that 

only non particle-associated mRNA (but not total 

mRNA) was found to correlate with the clinical 

status of the patient (21). 

Heating RNAs at 65 °C for 5 min followed by 

immediate placement on ice, as already suggested 

by some authors (22, 23), significantly improved 

mRNAs transcription to cDNAs. One 

possible explanation could be the enhancement 

of annealing of random primers to the RNAs as 

a consequence of the removal of any secondary 

structures. 

The quantification was most reliable with the use 

of 5’-end amplicons of the transcript. This is supported 

by a few of the relevant publications. It 

accords with the finding of Wong et al. that placental 

mRNAs in maternal plasma are predominantly 

5’-end fragments rather than intact transcripts 

(16). It also accords with the current belief 

that mRNA decay proceeds mostly from the 3’end 

(24, 25). Furthermore, 25 % of the expressed 

genes have a poly (A) tail of less than 30 residues 

in a significant proportion of their transcripts (26). 

However, the influence of amplicon target site selection 

on plasma mRNA quantification depends 

substantially also on the clinical status of the 

investigated subject. For instance, in our healthy 

subjects and statin-treated SA patients 5’-end am- 

Figure 2. Time dependent decrease of plasma MMP9 mRNA 

level (exon boundary 1-2; standardised to plasma PGK1 

mRNA level) in EDTA whole blood stored at 4 °C* 

*MMP9, gene encoding matrix metallopeptidase 9; PGK1, gene 

encoding phosphoglycerate kinase 1.

plicons gave the most reliable measurements and 

3’-end amplicons usually gave low plasma levels 

and low detection rates. In contrast, in unstable 

clinical conditions (patients with AMI), middle 

amplicons gave surprisingly low mRNA levels. 

Such particular differences should be evaluated 

and taken into account in every study design. 

Our study has shown the possibility of plasma 

mRNA analysis from genes responsible for atherosclerotic 

plaque development and destabilization 

and further implicate that clinical evaluation 

of the new analytical approach is reasonable. Plasma 

nucleic acids analysis is an emerging field for 

non-invasive in vivo assessment and monitoring 

of pathological processes in tissues of their origin. 

It is an alternative or supplement to protein 

analysis. Thus, plasma mRNA analysis may offer 

non-invasive in vivo assessment and monitoring 

of gene expression profile in vascular beds. It 

may offer non-invasive early diagnosis of vulnerable 

atherosclerotic coronary plaques and thus 

of patients who could suffer from acute coronary 

syndrome. Beside clinical evaluation of the new 

analytical approach further method optimisation 

is reasonable. We believe that with the use of higher 

plasma volumes for nucleic acids isolation 

(commercial kits are available) and with further 

improvement of the molecular methods there 

will be possible to quantify broader spectrum of 

mRNAs from genes involved in atherosclerotic 

plaque development and destabilization. 

We suggest that for plasma mRNA analysis 

EDTA whole blood samples should be centrifuged 

within one hour of venesection. There are 

rare studies of mRNA stability after venesection 

and results are contradictory (13, 23). Plasma 

mRNA stability may depend on the measured 

transcript and on the method of transcript quantification 

(RNA isolation and transcription to 

cDNA, selection of amplicon target site and PCR 

quantification, to name only some determinants). 

By using our optimized method we were not able 

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to ignore the requirement for centrifugation of 

EDTA blood immediately after venesection. Namely, 

mRNA stability in EDTA blood appears to 

be much more dependent on the clinical status 

of the investigated subject than on the selected 

transcript or on the method for its quantification. 

In general, the decay rate was higher in healthy 

volunteers and patients with SA with statin treatment 

than in patient with AMI. One reasonable 

explanation could be that plasma mRNAs in acute 

coronary syndrome are predominantly proteinassociated 

and more resistant to degradation and, 

in stable condition, predominantly non particleassociated 

and thus more exposed to degradation. 

Finally, it was sometimes observed that a sample 

processed immediately after venesection (within 

few minutes) yielded very low plasma mRNA levels, 

which was not investigated in details and is 

not explained in pertinent literature. 

Limitation of our study is that the methods were 

developed and optimised with the use of relatively 

small number of plasma samples. Further 

evaluations in larger cohorts and in real clinical 

settings are needed. Furthermore, biologic variability 

of plasma mRNAs and potential endogenous 

and exogenous analytical interferences (i.e. 

statins) should be evaluated. 

In conclusion, we are the first showing that in patients 

with coronary atherosclerosis, as well as in 

healthy subjects, it is possible to reliably quantify 

plasma mRNA level from genes responsible for 

plaque development and destabilization. Plasma 

mRNA estimation potentially offers non-invasive 

in vivo assessment and monitoring of gene expression 

profile in vascular beds. 

ACKNOWLEDGMENTS/ DISCLOSURES 

The authors acknowledge financial support from 

the Slovenian Research Agency (project No. L3- 

0129-2413-08). The project was also financially 

supported by Dade Behring Austria GmbH. 


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Cystatin C in sera of patients with aggressive non-Hodgkin B-cell 

lymphoma 

Adaleta Softić 1 , Lejla Begić 1 , Alma Halilbašić 2 , Janko Kos 3 

1 2 Faculty of Pharmacy, University of Tuzla, Department of Hematology, University Clinical Centre; Tuzla, Bosnia and Herzegovina, 

3Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia 


Adaleta Softić 

Faculty of Pharmacy, University of Tuzla 

Univerzitetska 8, 75000 Tuzla, 


Phone: +387 35 320 611; 

fax: +387 35 320 601 

E-mail: adaleta_m@yahoo.com 





Accepted: 


ABSTRACT 


Aim To investigate the cystatin C levels in sera of patients with 

aggressive non-Hodgkin B-cell lymphoma. 

Methods The levels of cystatin C in sera of lymphoma patients 

and control group consisted of healthy individuals, were measured 

by using specific sandwich-type ELISA. For each patient the 

clinical stage of disease was determined according to Ann Arbor 

staging system for lymphomas. 

Results Our study shows that mean cystatin C serum level in the 

patients group (1056 ± 65 ng/mL) was significantly higher when 

compared with the mean level of the healthy control group (819 

± 28 ng/mL) (P=0,001). Mean cystatin C level of the group with 

clinical stages III and IV (1255 ± 109 ng/mL) was significantly 

elevated when compared with the mean level of the group with 

clinical stages I and II (896 ± 51 ng/mL) (P=0,03). 

Conclusion This finding points out a connection between inhibitor 

level and aggressive behaviour of lymphoma and could be considered 

for further strategies of prognosis of the disease. 

Key words: cystatin C, non-Hodgkin B-cell lymphoma 

97

98 


INTRODUCTION 

Cystatin C is a non-glycosilated 13 kDa protein inhibitor 

of cysteine proteases, with widespread distribution 

in bodily fluids, the highest levels having 

been determined in cerebrospinal fluid, seminal 

plasma, and sinovial fluid (1). A broad spectrum of 

biological roles have been suggested for cystatin 

C, including control of protein catabolism, regulation 

of hormone processing and bone resorption, 

inflammation, antigen presentation and T-cell dependent 

immune response (2,3). 

Since cystatin C is produced at a constant rate by 

all nucleated body cells, freely filtered in the renal 

glomeruli and almost completely reabsorbed 

and catabolised in the proximal tubules, it was 

suggested as a marker of glomerular filtration 

rate (4). 

Cystatin C has also been suggested as playing a 

role associated with alteration of the proteolytic 

system in cancer. In patients with colorectal, lung 

and melanoma carcinomas high serum levels of 

cystatin C are associated with poor outcome of 

disease (5-7). 

In order to extend the knowledge about cystatin 

C function in malignant diseases, we studied the 

cystatin C levels in sera of patients with aggressive 

non-Hodgkin B-cell lymphomas. 


Serum samples have been collected for two years 

at the Department of Hematology of the University 

Clinical Centre of Tuzla. Samples were obtained 

from 27 patients with clinically approved aggressive 

non-Hodgkin B cell lymphomas, who were 

receiving chemotherapy. Twenty-six patients had 

diffuse large cell lymphoma and one patient had 

a third grade follicular lymphoma. Patients with 

renal disfunction, autoimmune diseases, asthmatic 

patients and other malignancies were excluded 

from the study. For each patient a clinical stage 

of disease was determined according to Ann Arbor 

staging system for lymphomas (8,9). For 17 

patients creatinine levels were noted, at the time 

serum samples were collected. A control group of 

sera consisted of 33 healthy blood donors. 

Five milliliters of blood per patient or donor have 

been collected. The blood was clotted at 4-8 ºC 

and subsequently centrifuged at 3000 rpm. Serum 

was separated, aliquoted and stored at -20 

ºC until analysis. 

The levels of cystatin C in sera of lymphoma patients 

and healthy individuals were measured by 

using specific sandwich-type ELISA, developed 

at the Department of Biochemistry and Molecular 

Biology, Jožef Stefan Institute and Krka, d.d., 

as described (7). A microtiter plate was coated 

with rabbit affinity purified anti-cystatin C polyclonal 

antibody. Recombinant human cystatin C 

was used for preparation of calibration curves. 

Sera from patients and blood donors were diluted 

1:80 prior to being applied to wells of microtiter 

plate. As a detection antibody, 1A2 monoclonal 

antibody was used, conjugated to horseradish peroxidase. 

A substrate for peroxidase was 3,3΄,5,5΄tetramethylbenzidine 

liquid substrate system 

(TMB, Sigma). The reaction was stopped by additing 

of 2M H SO , and the absorbance was me- 

2 4 

asured at 450 nm on minireader (SLT Rainbow; 

Salzburg, Austria). Measured values of diluted 

samples and controls were compared with the calibration 

curve and expressed in g/L of serum. 

Serum creatinine level was determined using kinetic 

Jaffe method according to the manufacturer’s 

procedure (Boehringer, Mannheim). 

Differences of mean cystatin C levels between 

the patients group and the control, and inside patients 

group were tested by non-parametric Mann-Whitney 

test. Correlation of cystatin C values 

with serum creatinine was tested by non-parametric 

Spearman rank correlation coefficient test. In 

all tests, two sided P values below 5% were considered 

significant. 

RESULTS 

Cystatin C was significantly elevated (mean 

1056 ± SE 65ng/mL) in the patient population 

when compared to control levels (mean 819 ± 

SE 28 ng/mL) (Table 1) (P=0.001). The inhibitor 

levels were correlated positively with creatinine 

levels, with Spearman correlation coefficient 

r=0.48 (P=0.05) 

Patients were staged according to the Ann Arbor 

staging system. Mean cystatin C level of stages 

Table 1. The mean levels of cystatin C in sera of patients with 

aggressive non Hodgkin B- cell lymphoma and in healthy 

control* 

Group 

Lymphoma 

patients 

Number of 

persons 

Cystatin C sera level (x 10 -6 g/L) 

Mean ± SE 

27 1056 ± 65 

Control 33 819 ± 28 

*SE, standard error of mean

III and IV (1255 ± SE 109 ng/mL) was compared 

with mean level of stages I and II (896 ± SE 51 

ng/mL) (Table 2). The inhibitor was significantly 

elevated in stages III and IV (P=0.03). 

DISCUSSION 

Although inflammatory conditions and malignancy 

have been reported not to affect circulating 

cystatin C, in several clinical studies on melanoma, 

colorectal and hepatocellular carcinoma, 

a significant increase in cystatin C level was found 

in malignant sera (5,7,11). It has been debated 

whether it is possible that cystatin C is also 

upregulated in cancer, as it appears to be the case 

with cathepsins (12,13), or whether the alteration 

in glomerular filtration rate induces the increase 

in cystatin C sera level in cancer patients (14). 

The present study shows that serum cystatin C 

is significantly elevated in patients with aggressive 

non-Hodgkin B cell lymphoma compared to 

controls, which corresponds to the observations 

made in other types of cancers (5-7,15). 

To exclude the possibility that high levels of the 

inhibitor are the result of renal cell dysfunction, 

we noted and analyzed patients’ creatinine level. 

Only one patient had the creatinine value above 

the upper physiological limit, 105 μmol/L. The 

obtained values for creatinine were correlated 

with cystatin C levels. Although statistically 

significant, correlation coefficient (r=0.48) is 

much lower compared to the correlation coefficient 

in healthy population (r=0.67) (10). This 

result supports the idea that the elevated cystatin 

C levels were not only the result of the renal cell 

disfunction and that there are some other factors 

which, in this type of malignancy, lead to higher 

expression of cystatin C gene or higher secretion 

of the inhibitor into body fluids. 

According to the Ann Arbor staging system, stages 

I and II indicate that lymphoma infiltrates one or 

two groups of lymph nodes which are located at 

Table 2. Patients grouped according to Ann Arbor staging 

system* 

Ann Arbor stage 

of disease 

Number of 

patients 

Mean cystatin C 

level ± SE 

(x 10 -6 g/L) 

I II III IV 

1 14 7 5 

I , II 

896 ± 51 

III , IV 

1255 ± 109 

*Mean inhibitor level and standard error (SE) for groups I, II and 

III, IV 

Softić et al Cystatin C in non-Hodgkin lymphoma 

one side of the diaphragm. Stages III and IV indicate 

that the lymphoma has spread to both sides of the 

diaphragm with (stage IV) or without (stage III) involvement 

of extranodal organ(s) like bone marrow 

or liver (9). In diffuse large cell lymphoma the level 

of cystatin C is significantly elevated in group III 

and IV when compared to group I and II. This finding 

may point to a possible connection between 

inhibitor level and aggressive behaviour of this 

group of lymphoma. This result is in line with results 

of other researches, according to which at the 

systemic level, cystatin C seems to promote tumour 

progression, presumably due to its multifunctional 

properties, regulating processes such as immune 

response, apoptosis, or cell adhesion (16,17). 

Corticosteroids, which are included in therapeutic 

protocols for lymphoma patients, have also 

been suggested to contribute to higher cystatin 

C serum levels. For example, cystatin C was 

significantly increased in steroid-independent 

asthmatic patients after one-week treatment with 

methylprednisolone (18). The effect of corticosteroids 

could also explain the increased cystatin 

C levels in myeloma patients (19). As reported 

by Bjornadottir et al. (20), glucocorticoid increased 

promotor-mediated transcription of cystatin 

C gene in HeLa cells, resulting in higher cystatin 

C expression and secretion. However, the effect 

of corticosteroids can not explain significantly 

higher cystatin C values in sera of patients within 

group III and IV when compared to group I and 

II. Unfortunately, in this study we did not investigate 

the effects of type and number of chemotherapeutic 

protocols on cystatin C level. These factors 

are the subject of our further research work. 


This study was partly presented as poster presentation 

by Softć A, Begić L, Halilbašić A, Kos 

J. Cystatin C in sera of patients with aggressive 

non-Hodgkin B-cell lymphoma. Proceedings of 

the 1st Congress of Medical Biochemists of Bosnia 

and Herzegovina With International Participation, 

Sarajevo/Bosnia and Herzegovina, May 

21-22 2010. Programme & Abstracts, Association 

of Medical Biochemists in Bosnia and Herzegovina, 

Sarajevo, 2010, p 44. 

The work was supported by the Research Agency 

of the Republic of Slovenia. 


99

100 


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6. Zore I, Krašovec M, Cimerman N, Kuhelj R, Werle 

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Kos J, Werle B, Lah T, Brunner N. Cysteine protei- 

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Štabuc B, Vrhovec L, Štabuc-Šilih M, Cizej TE. 

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by serum cystatin C: use in cancer patients 

before and during chemotherapy. Clin Chem 2000; 

46:193-7. 

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Cistatin C u serumu pacijenata s agresivnim ne-Hodgkinovim limfomom B ćelija 

Adaleta Softić1 , Lejla Begić1 , Alma Halilbašić2 , Janko Kos3 1 2 3 Farmaceutski fakultet, Univerzitet u Tuzli; Hematološko odjeljenje, Univerzitetski klinički centar Tuzla, Bosna i Hercegovina; Farmaceutski 

fakultet, Univerzitet u Ljubljani, Ljubljana, Slovenija 

SAŽETAK 

Cilj Analizirati nivo cistatina C u serumima pacijenata s agresivnim ne-Hodgkinovim limfomom B 

ćelija. 

Metode Nivo cistatina C u serumima bolesnika i kontrolnoj skupini, sačinjenoj od zdravih osoba, mjeren 

je specifičnom ‘’sendvič’’ ELISA metodom. Za svakog pacijenta klinički stadij bolesti određen je 

prema Ann Arbor staging sistemu za limfome. 

Rezultati Naša studija je pokazala da je prosječni nivo cistatina C u serumu bio značajno povišen u 

pacijenata s agresivnim ne-Hodgkinovim limfomom B ćelija (1056 ± 65 ng/mL) u odnosu na zdravu 

skupinu ispitanika (819 ± 28 ng/mL) (P=0,001). Prosječna vrijednost cistatina C u grupi pacijenata s 

kliničkim stadijem III i IV (1255 ± 109 ng/mL) bila je značajno viša u odnosu na prosječnu vrijednost 

inhibitora u pacijenata s kliničkim stadijem I i II (896 ± 51 ng/mL) (P=0,03). 

Zaključak Rezultati ukazuju na povezanost nivoa inhibitora i agresivnog ponašanja limfoma i mogu se 

uzeti u obzir prilikom kreiranja strategije za prognoziranje bolesti. 

Ključne riječi: cistatin C, ne-Hodgkinov limfom B ćelija. 

Original submission: 07 October 2010; Revised submission: 06 December 2010; Accepted: 08 December 2010.


Local CD4+, CD8+ and CD56+ reactions to lung cancer in regard 

to pathohistological type and clinical stage 

Edin Jusufovic 1,2 , Ermina Iljazovic 2,3 , Mitja Kosnik 4 , Dragan Keser 1,2 , Peter Korosec 4 , Edin Zukic 1 , Besim 

Prnjavorac 5,6 , Rifat Sejdinović 5 , Ekrem Ajanović 5 

1 Policlinic for Pulmonary Diseases, Medical Centre of Tuzla, 2 School of Medicine, University in Tuzla, 3 Department for Pathology, 

Policlinic for Laboratory Diagnostics, University Clinical Centre Tuzla; Tuzla, Bosnia and Herzegovina, 4 Department of Clinical Immunol- 

ogy and Molecular Genetics, University Clinic of Respiratory and Allergic Diseases Golnik, Golnik, Slovenia, 5 Department of Internal 

medicine, General Hospital Tesanj, Tešanj, 6 School of Pharmacy, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 


Edin Jusufovic 

Polyclinic for Pulmonary Diseases of 

the Medical Centre “Dr. Mustafa Sehovic” 

Albina Herljevica 1, 75 000 Tuzla, 


Phone: +387 35 281 711; 

Fax.: +387 35 282 161 

e-mail address: edinjusufovic@yahoo.de 





Accepted: 


ABSTRACT 


Aim To determinate the difference of abundance of CD4+, CD8+ 

and CD56+ bronchoalveolar fluid’s lymphocytes and their subpopulations 

between non- and small cell lung cancer. Also, the differences 

of abundance of examined lymphocytes were compared 

between main clinical stages of lung cancer. 

Methods Mini-bronchoalveolar lavate was taken from lungs of 55 

patients with cancer. After laboratory processing and adding CD3, 

CD4, CD8, CD27, CD28 and CD56 antibody, the material was 

analysed by flow cytometer. Results of Mini-BAL for non- and 

small cell lung cancer were compared, as well as the different clinical 

stages of the disease. 

Results Immature and regulatory forms of lymphocytes are more 

activated, while mature and activated forms are less activated in 

small cell lung cancer compared to non small type. With an increase 

of the clinical stage of disease, immunological reaction of T 

lymphocytes is better expressed because of increasing of abundance 

of immature and regulatory forms of different subpopulations 

of lymphocytes. 

Conclusion All components of local CD4+ and CD8+ T lymphocyte, 

as well as NK and NKT cells response were more activated 

in lungs with small cell lung cancer, and these reactions were more 

expressed with an increase in the clinical stage. 

Key words: Lung cancer, bronchoalveolar lavate, C4+ T-lymphocytes, 

C8+ T-lymphocytes, C56+ lymphocytes 

101

102 


INTRODUCTION 

Lung cancer shows greater mortality than cancers 

of breast, prostate, colon and pancreas together 

(1). Because of the high case-fatality rate of lung 

cancer, the incidence and mortality rates are nearly 

equivalent. Men are still significantly more likely 

to suffer from lung cancer than women (2). 

There are several types of antigens on the tumorous 

cells such as NY-ESO-1 (cancer-testis antigen), 

WT1 (Wilms tumor 1 antigen), MAGE family 

(MicroArray and Gene Expression), gp100 

(antigen melanoma-associated antigen), SART-1 

(squamous cell carcinoma antigen recognized by 

T cells), tirosinases, MUC 1 (cell surface associated 

antigen, mucin 1), MRP3 (ATP-binding 

cassette transporter) etc (3, 4). These antigens are 

believed to be crucial in the activation of numerous 

immune reactions to the tumor, including 

T lymphocyte reaction that is believed to have 

a central role in antitumour immunity. T lymphocytes 

are a numerous, but heterogeneous population 

and have regulatory as well as effector 

functions (5). 

CD4+ T lymphocytes (CD – engl. cluster of differentiation) 

are activated by antigen presenting 

cells and posses a helper function and are activated 

by antigen presenting cells. These lymphocytes 

also show a significant immunoregulatory 

activity (6). CD8+ T lymphocytes are effector 

cells with cytolytic and cytotoxic function (5, 7). 

Currently there is only a limited number of studies 

that address the phenotypic classification of 

human CD4+ and CD8+ T lymphocytes. On the 

other hand the process of their differentiation is 

quite well established. We know that CD27+28+ 

subpopulations are immature memory cells, and 

CD27-CD28- subtype are mature forms of CD4+ 

and CD8+ T lymphocytes (5, 7, 8). NK (NK – 

engl. natural killer) cells are lymphocytes with 

particularly cytolytic activity, capable of spontaneously 

destroying tumor cells and even metastatic 

tumor cells in the systemic circulation (9). 

NKT cells (NKT – engl. natural killer T) represent 

0.2% of all T-cells in the peripheral blood, 

and are characterized by swift and abundant secretion 

of cytokines (IL-2, IFNγ, TNFα and IL-4) 

(10, 11). NK and NKT cells are of CD56+ type. 

All CD56+ cells are further classified to CD4+ 

and CD8+, each one having a different biological 

base (9, 10, 11). 

There are five basic pathohistological types of 

lung cancer: adenocarcinoma, squamous cell 

carcinoma, large cell carcinoma, small cell carcinoma 

and bronchioalveolar carcinoma (12). 

Among these, small cell carcinoma shows the 

most malignant and aggressive behaviour (13, 

14). This was the reason of establishing a new 

lung cancer classification, which divide this disease 

in only two major groups: small cell lung 

cancer - SCLC and non-small cell lung cancer 

- NSCLC (13). 

TNM (TNM – engl. Tumor, Nodus, Metastasis) 

system is a system for lung cancer staging. the 

7th edition of TNM staging in lung cancer applies 

equally well to all histologies of NSCLC, but 

TNM for SCLC is less helpful. Most patients 

have advanced disease at the time of presentation 

(15). Currently, worldwide including Europe, 

in the very time of the diagnosis almost 50% of 

patients with lung cancer are in the clinical stage 

III or IV (1). 

Lung cancer represents a diagnostic, especially 

early-diagnostic, therapeutic, and public-health 

challenge. Therefore, research in this field 

is essential. It is believed that the basic cytological, 

cytogenetic, molecular, immunogenetic 

and immunological studies provide data for the 

fundamental understanding of this problem, also 

different modalities of therapeutic approaches. 

Among many prognostic markers immunological 

parameters seem to have a very important role 

(16). Currently, we are at the beginning of a new 

classification of lung cancer considering immunological 

features of five basic pathohistological 

types of this carcinoma (3). 

The aim of this study was to determinate the difference 

in CD4+, CD8+ and CD56+ local immunological 

reaction with regard to pathohistological 

type and clinical stage of lung cancer. 


This study was designed as a cross-sectional investigation. 

All laboratory analyses were completely 

done in the Department of Clinical Immunology 

and Molecular Genetics at University Clinic 

of Respiratory and Allergic Diseases of Golnik, 

Slovenia. 55 patients were included in study of 

CD4+ and CD8+ lymphocyte, and 46 patients in 

study of CD56+ lymphocyte. The inclusion criterion 

was the pathohistologically confirmed dia-

gnosis of primary lung cancer. Exclusion criteria 

were previous chemotherapy, radiotherapy or other 

anti-cancer therapy, absence of lymphocytes 

in the cytological samples of the tumor, non-representative 

number of events1 in flow-cytometric 

analyses of the material (lower than 80 for 

lymphocytes in mini-BAL2 ). 

All patients had a mini bronhoalveolar lavage 

(mini-BAL) taken during a routine diagnostic 

flexible bronchoscopy. During the procedure 20 

ml of 0.9% NaCl solution was instilled. Mini- 

BAL was taken from the cancerous lung and from 

the healthy lung of the same patient. The material 

obtained with the mini BAL was centrifuged 

at 1600 rpm, the supernatant was poured out, and 

0.5mL of azid Hemacel-a solution was added followed 

by 1 ml of solution for deep freezing. The 

material was frozen at -70 ºC and preserved until 

further processing. 

Flow cytometer was used to count the lymphocytes 

in 100 μl samples. The number of lymphocytes 

and the percentage of T-lymphocyte subpopulations 

in the mini-BAL was determined, including 

the immature (CD27+28+), mature (CD27-28-) 

forms of CD4+ and CD8+ T lymphocytes, also 

the analysis included the regulatory (CD4+) and 

activated (CD8+) NK and NKT cells. 

The material obtained using miniBAL was stained 

with monoclonal antibodies and prepared 

for flow cytometry using the procedural technique 

described by the manufacturer. Following 

antibodies were used: CD4-FITC: BD-2010; 

CD8-APC: BD-2010; CD27-PE: BD Pharmigen- 

2014, CD28-Cy-Chrome: BD Pharmigen-2010 

and CD56-PE: IQ Products 2010. The prepared 

material was then processed by the flow cytometer 

FACS Calibur basic G4, manufactured in 

2001 by Becton Dickinson. The following parameters 

were used: FSC-H/SSC-H for lymp- 

Figure 1. Number of helper lymphocytes in mini-BAL with 

regard to pathohistological type of lung cancer 

Jusufović et al T cell reaction on lung caner 

hocytes, FL4-H/SSC-H for CD4+ lymphocytes 

and FL1-H/SSC for CD8+ lymphocytes, FL2-H/ 

FL3-H for CD27/CD28, FL3-H/FL1-H for CD3/ 

CD8, FL3/FL4-H for CD3/CD4, FL1-H/FL2-H 

for CD8/CD56 and FL4-H/FL2-H for analyzing 

CD4/CD56. 

In the analyses we used nonparametric and parametric 

methods for calculating statistical significance. 

Mann-Whitney test, Student’s t-test, χ2 test, Fisher test were used to calculate differences 

between the groups. Kruskal-Wallisov test was 

used to analise the variance in the group. 

Statistical hypotheses were tested with the level 

of α=0.05, in other words, the difference between 

groups in the sample was considered significant if 

p

104 


Figure 3. Number of NK cells and NKT cells in mini-BAL with 

regard to pathohistological type of lung cancer 

The number of CD4+ (Figure 1) and CD8+ (Figure 

2) T lymphocytes was statistically higher 

(p

Figure 7. Number of helper lymphocytes in mini-BAL with 

regard to clinical stage of lung cancer 

(p

106 


Figure 11. Mini-BAL abundance of mature and immature 

subpopulations of helper lymphocytes with regard to clinical 

stage of lung cancer 

In this study we have concluded that immature 

and regulatory lymphocyte’s components of immunological 

reaction on lung cancer were more 

excitated in small cell type lung cancer comparing 

to other pathohistological types, whereas the 

mature and activated T cells are less enhanced in 

this type of lung cancer. Chansac et all. 2005 (18) 

have published similar results. They showed that 

the loss of MHC class I molecules allows these 

tumor cells to avoid the inevitable destruction, 

mediated by immune competent cells primarily 

by CD8+T lymphocytes. 

Our study showed that with the increase of the 

clinical stage of primary lung cancer the mean 

number of CD4+, CD8+, NK and NKT lymphocytes 

events in BAL increased. These results 

correspond to the aforementioned theoretical 

debates about the characteristics of the immune 

response to cancer, claiming that the increasing 

level of clinical stage of tumor correlates to the 

degree of infiltration of the appropriate clones of 

immune cells. Numerous studies have reported 

on a correlation between the number of tumor- 

Figure 12. Mini-BAL abundance of mature and immature subpopulations 

of cytotoxic T lymphocytes with regard to clinical 


Figure 13. Mini-BAL abundance of regulatory and activated 

subpopulations of NK and NKT cells with regard to clinical 


infiltrating lymphocytes in histologically analyzed 

lung cancer tissue with pathologic grade of 

the disease, tumor size and vascular invasion and 

survival (16). 

In this study we also concluded that with the increase 

of the clinical stage of lung cancer, the 

excitation of local lymphocyte reaction became 

more pronounced with predominant abundance 

of effectory insufficient helper and cytotoxic 

lymphocytes, as well as NK and NKT cells. As 

previously discussed, as the malignant process 

advances, there is an increase only in the quantitative 

characteristics of T-cell immune response, 

at the expense of immature or regulatory phase. 

Since we were unable to find similar studies related 

to lung cancer in the available literature, and 

compare them with our results, additional research 

is required. However we may say that these 

results already provide evidence to the theory of 

immune surveillance against cancer and the role 

of individual components of the immune response 

to malignant process. 

This study has some limitations. The first arises 

from the fact that during the mini-BAL procedure 

volume obtained was not measured, and the cells 

were concentrated only in the volume of one 

specimen (test tube). So the samples of cells concentrated 

to 1mL from different amounts of BAL 

were compared. However, if we assume that the 

sample of 55 patients is large enough, and that 

these errors are equally distributed in samples 

of healthy and diseased lungs, then the results 

of this study may be accepted while consciously 

taking into account certain level of relative error. 

The second limitation resumes that this study did 

not take into account the level of influence that 

postobstructional pneumonitis had on local im-

mune response. The third limitation is a substantial 

possibility of systemic effects of tumor on the 

diseased lung. However, if we assume that the 

systemic effects bring the same changes to the 

healthy and affected lungs, then we could compare 

them and attribute the difference to the local 

reaction to malignancy. The fourth limitation of 

this studyis that it did not analyze the function of 

lymphocytes, but only their surface markers. 

In spite of these limitations, this study, which is 

primarily of basic nature, suggests a new perspective 

of the cancerous process of lungs and 

poses a realistic basis for further research in this 

direction. 

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Special acknowledgement to Aleš Rozman, MD, 

Master Degree, University Clinic of Respiratory 

and Allergic Diseases of Golnik, Slovenia, who 

collected all cytological material indispensable 

for this study. 

Professor Stahov Jugoslav, PhD, School of Science 

of the University in Tuzla, Bosnia and Herzegovina, 

did all statistical analyses and graph 

designs. 

Competing interests: none to declare. 

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J, Pack M, Hutchinson A, Geller M, Liu N, 

Annable R, Shay J, Kirchhoff K, Nishi N, Ando Y, 

Hayashi K, Hassoun H, Steinman RM, Dhodapkar 

MV. Sustained expansion of NKT cells and antigen-specific 

T cells after injection of α-galactosylceramide 

loaded mature dendritic cells in cancer 

patients. J Exp Med 2005; 201:1503–7. 

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cells: Roles in Tumor Immunosurveillance and Tolerance. 

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A. Changes in the distribution of lung cancer 

cell types and patient demography in a developing 

multiracial Asian country: Experience of a university 

teaching hospital. Lung Cancer 2006; 53:23-30. 

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Lung, Larynx, and Pleura. In: Adami H, Hunter D, 

Trichopoulos D, eds. Textbook of Cancer Epidemiology. 

2nd Edition. Oxford: Oxford University Press, 

2008; 349-67. 

14. Hiraoka K, Miyamoto M, Cho Y, Suzuoki M, Oshikiri 

T, Nakakubo Y, Itoh T, Ohbuchi T, Kondo S, 

Katoh H. Concurrent infiltration by CD8+ T cells 

and CD4+ T cells is a favourable prognostic factor 

in non-small-cell lung carcinoma. Br j Cancer 2006; 

94:275-80. 

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TNM staging system for lung cancer. Ann Thorac 

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Macri L, Daniele L, Oliaro A. Clinical significance 

of tumor-infiltrating lymphocytes in lung neoplasms. 

Ann Thorac Surg 2009; 87:365-72. 

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T, Aokage K, Saijo N, Nishiwaki Y, Gemma 

A, Kudoh S, Ochiai A. Predominant infiltration of 

macrophages and CD8+ T cells in cancer nests is a 

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cell lung cancer. Cancer 2008; 113:1387-95. 

107

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18. Chansac BlM, Moretta A, Vergnon I, Opolon P, Lécluse 

Y, Grunenwald D, Kubin M, Soria JC, Chouaib, 

Mami-Chouaib F. NK Cells Infiltrating a MHC Class 

I-Deficient Lung Adenocarcinoma Display Impaired 

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Associated with Altered NK Cell-Triggering Receptors. 

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Huang ZX, Yang HB, Wu C. CD4+CD25+ regulatory 

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Medicine 2005; 172:1434-9. 

Lokalna CD4+, CD8+ i CD56+ reakcija na karcinom pluća u zavisnosti od 

patohistološkog tipa i kliničkog stadija oboljenja 

Edin Jusufovic1,2 , Ermina Iljazovic2,3 , Mitja Kosnik4 , Dragan Keser1,2 , Peter Korosec4 , Edin Zukic1 , Besim 

Prnjavorac5,6 , Rifat Sejdinović5 , Ekrem Ajanović5 1 2 3 Poliklinika za plućne bolesti, Doma zdravlja Tuzla; Medicinski fakultet, Univerzitet u Tuzli; Zavod za patologiju, Poliklinika za laboratorijsku 

dijagnostiku, Univerzitetski klinički centar Tuzla, Tuzla, Bosna i Hercegovina; 4Odjeljenje za kliničku imunologiju i molekularnu 

medicinu, Univerzitetska bolnica za plućna oboljenja i alergije Golnik, Golnik, Slovenija; 5Odjeljenje za internu medicinu, Opšta bolnica 

Tešanj, Tešanj; 6Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo, Bosna i Hercegovina 

SAŽETAK 

20. Janssen-Heijnen ML, Coebergh JW, Klinkhamer 

PJ, Schipper RM, Splinter TA, Mooi WJ. Is there a 

common etiology for the rising incidence of and decreasing 

survival with adenocarcinoma of the lung? 

Epidemiology 2001; 12:256-8. 

21. Ikeda S, Funakoshi N, Inagaki M, Shibata T. Clinicopathologic 

roles of tumor-infiltrating lymphocytes 

and CD8-positive lymphocytes in lung cancer imprint 

smears in squamous cell carcinoma and adenocarcinoma. 

Acta Cytol 2006; 50:423-9. 

Cilj Utvrditi razliku u zastupljenosti CD4+, CD8+ i CD56+ limfocita i njihovih subpopulacija u bronhoalveolarnom 

lavatu pluća kod ne- i mikrocelularnih karcinoma pluća, te odnos zastupljenosti navedenih 

limfocitnih subpopulacija i visine kliničkog stadija oboljenja. 

Metode Uzet je mini-bronhoalveolarni lavat od 55 bolesnika iz pluća zahvaćenog karcinomom. Materijal 

je analiziran protočnim citometrom, nakon dodavanja CD3, CD4, CD8, CD27, CD28 i CD56 antitijela. 

Poredili su se rezultati mini-BAL-a ne- i mikrocelularnih karcinoma pluća i različitih kliničkih 

stadija ovog oboljenja. 

Rezultati Kod mikrocelularnog karcinoma pluća, nezrele i regulatorne komponente T-limfocitnog imunološkog 

odgovora značajno su više podražene, dok su zrele i aktivirane forme manje podražene u 

odnosu na ostale patohistološke tipove ovog oboljenja. S porastom visine kliničkog stadija karcinoma 

pluća, podraženost T-limfocitnog imunološkog odgovora postaje izraženija na račun zastupljenosti nezrelih 

i regulatornih formi pojedinih subpopulacija T-limfocita. 

Zaključak Sve komponente lokalnog CD4+ i CD8+ T limfocitnog, te NK i NKT imunološkog odgovora, 

značajno su više podražene kod mikrocelularnog karcinoma pluća. Ove reakcije postaju više 

podražene s porastom kliničkog stadija oboljenja. 

Ključne riječi: karcinom pluća, bronhoalveolarni lavat, CD4+ limfociti, CD8+ limfociti, C56+ limfociti. 



Interleukin 18 expression in the primary breast cancer tumour 

tissue 

Nahida Srabović 1 , Zlata Mujagić 1 , Jasminka Mujanović-Mustedanagić 2 , Zdeno Muminović 2 , Elmir 

Čičkušić 2 

1 2 Department of Biochemistry, School of Pharmacy, University of Tuzla, Department of Laboratory Diagnostics, University Clinical 

Centre of Tuzla; Tuzla, Bosnia and Herzegovina 


Nahida Srabović 

Faculty of Pharmacy, University of Tuzla 

Univerzitetska 8, 75 000 Tuzla, 


Phone: +387 35 320 990; 

fax.: +387 35 320 991 

E-mail: nahida.srabovic@gmail.com 





Accepted: 


Abstract 


Aim To investigate the presence and expression levels of the IL- 

18 in the primary breast cancer tissue in relation to the unchanged 

breast tissue in same patients and the breast tissue in patients with 

benign breast disease, as well as the correlation between the IL- 

18 expression levels and pathohistological factors, including the 

correlation between IL-18 expression and the estrogens and progesterone 

receptor status. 

Methods This prospective randomized study was conducted at the 

Policlinic for Laboratory Diagnostics of the University Clinical 

Centre of Tuzla. 50 patients with invasive ductal breast cancer and 

20 patients with benign breast diseases were included in the study. 

The tree-step immunohistochemical staining was used for testing 

the levels of IL-18 expression and hormone receptor status. 

Results IL-18 was present in the breast cancer tumour, in the surrounding 

unchanged tissue of the same patients and in the breast tissue 

of patients with benign breast tumour and other benign breast 

disease. The expression of this interleukin was significantly higher 

in breast cancer tumour tissue as compared to its expression in 

surrounding unchanged tissue of the same patients (p

110 


Introduction 

Breast cancer is the most common malignant tumour 

in women, accounting for approximately 

one third of all female cancers (1). The epithelial 

tumour microenvironment is made of complex 

tissue comprising variable numbers of tumour 

cells, fibroblasts, endothelial cells and infiltrating 

lymphocytes. Cytokines are key molecules 

controlling autocrine or paracrine communications 

within and between these individual cell 

types. Under some circumstances endogenous 

cytokines may control host responses against 

tumour, but there is increasing evidence that the 

cytokine network contributes to tumour growth, 

progression and host immune-suppression (2, 3). 

Cytokines, such as transforming growth factor–β 

(TGF–β), tumour necrosis factor–α (TNF-α), 

interleukins IL-1, IL-6, IL-10, IL-12, IL-18 and 

the interferons, all play a key role in controlling 

the immune response. Changes in cytokine levels 

mediated by the tumour, both directly and indirectly, 

are important parameters that affect the 

course of disease (1). 

Interleukin–18 (IL-18), originally known as 

interferon-γ (IFN-γ)–inducing factor, is a cytokine 

that shares structural and functional properties 

with IL-1. This cytokine is mainly produced by 

activated macrophages and dendritic cells (4). 

IL-18 has multiple biological activities via its 

capacity to stimulate innate immunity and both 

Th1 and Th2 mediated responses (5, 6). It exerts 

anti-tumour effect by enhancement of NK cell 

activity, reduction of tumorigenesis, induction of 

apoptosis and inhibition of angiogenesis in tumour 

cells (7, 8). In addition, recent studies have implicated 

its contribution in the pathogenesis and 

clinical outcome in patients with various cancers 

(4). Recent clinical studies associated this marker 

with prognosis in patients with gastric carcinoma, 

haematological malignancies (9) and breast cancer, 

especially metastatic breast cancer (10-12). 

It is also found that IL-18 is secreted by murine 

melanoma cells and this endogenous IL-18 is involved 

in the immune escape of murine melanoma 

cells (13). However, a few data exist related 

to in vivo presence of IL-18 in human tumours 

and there is a strong need to study its levels of 

expression in different types of tumours. 

The aim of this study was to determine the presence 

and the expression of the IL-18 levels in 

primary breast cancer tumour tissue in relation to 

its expression in unchanged breast tissue of the 

same patients and the breast tissue of the patients 

with benign breast disease, and also to investigate 

the correlation between the IL-18 expression 

levels and the pathohistological factors and 

between IL-18 expression and the estrogens and 

progesterone receptor status. 


Patients and tissue samples 

This study, approved by the Ethic Committee 

of the University Clinical Centre Tuzla, was a 

prospective randomised study with well defined 

cryteria for patients inclusion and exclusion. 

The inclusion criteria for patients were as follows: 

histologically proven invasive ductal breast cancer, 

no distance metastases, no previous adjuvant 

therapy, currently under no treatment, no other 

major illnesses. 

Tumour tissue samples and the samples of the 

surrounding unchanged tissue from patients with 

primary invasive ductal breast cancer were used, 

as well as breast tissue samples from patients 

with benign breast tumours and other benign breast 

diseases. 

A total of 70 patients was included in this study: 

24 with negative axillary lymph nodes, 26 with 

positive axillary lymph nodes and 20 with benign 

breast disease. 

Routine histological examination was performed 

with haematoxylin-eosin staining. All tumours 

were classified according to the criteria of the 

World Health Organization (14). Histological 

grade was obtained in accordance with a modified 

Scarff-Bloom-Richardson histological grading 

system. Staging was based on TNM (tumour-node-metastasis) 

system. Tumour size was 

evaluated separately (0,5-1 cm, ≤2 cm, 2-5 cm, 

>5 cm). 

Immunohistochemistry 

Immunohistochemical staining for oestrogen receptor 

(ER), progesterone receptor (PR) and IL- 

18 was performed on 4 µm thick formalin-fixed 

paraffin sections after the heating at 42°C in 0,2% 

BSA solution. Deparaffinisation and rehydration 

were performed in xylene and ethanol solutions

(reducing concentration 96% - 30%). Antigen 

retrieval was performed in the procedure with 

the retrieval buffer (pH=9,0, TRIS 20 mmol/L, 

EDTA 0,05mmol/L, 0,05 % Tween 20). In order 

to block non-specific antibody binding, normal 

goat serum and normal rabbit serum were applied. 

Subsequently, sections were incubated with 

the primary antibody and after rinsing with the 

PBS buffer, the secondary antibody was applied. 

A three-step technique was used for visualization 

with diaminobenzidine as a chromogen. Counterstaining 

was performed with hematoxylin. Slides 

were preserved in Canada balsam (turpentine). 

Primary antibodies dilution used in this study: 

a mouse antihuman monoclonal antibody against 

ER (clone NCL-ER-6F11; Visionbiosystem, 

Novocastra) 1:50 diluted in the PBS/BSA 

buffer pH=7,2; a mouse antihuman monoclonal 

antibody against PR (clone NCL-PGR-312; Visionbiosystem, 

Novocastra) 1:150 diluted in the 

PBS/BSA buffer pH=7,2; a goat polyclonal antihuman 

IL-18 (N-19; Santa Cruz Biotechnology, 

Inc., CA, USA) 1:50 diluted in the PBS/BSA buffer 

pH=7,2. 

Srabović et al IL-18 expression in the breast cancer tumour 

Secondary antibodies dilutions used: a goat antimouse 

polyclonal antibody biotin conjugated 

(Dako) 1:200 diluted in the PBS/BSA buffer 

pH=7,2; a rabbit antigoat polyclonal antibody 

biotin conjugated (sc-2774; Santa Cruz Biotechnology, 

Inc.CA, USA) 1:400 diluted in the PBS/ 

BSA buffer pH=7,2 

Immunohistochemical staining evaluation. The 

evaluation of the immunohistochemical staining 

was performed by a light microscopic observation 

(Olympus BX-50 light microscope). Remmeles 

immunoreactivity score (15) for ER- and PR 

– immunoreactivity was used for the evaluation 

of the results. 

The extent of IL-18 immunoreactivity was scored 

as 0 for absence of the positive cells, +1 for 

1% to 33% of positive cells, +2 for 34% to 66% 

of positive cells, and +3 for more than 66% of 

positive cells. The intensity of IL-18 immunoreactivity 

was scored as 0 for no staining, 1 for 

weak staining, 2 for moderate staining and 3 for 

strong staining. The extent and the intensity of 

IL-18 immunoreactivity score were observed separately. 

Table 1. Pathohistological factors, the intensity and the extent of IL-18 immunoreactivity in patients without lymph node metastasis* 

Intensity of IL-18 

Extent of IL-18 

immunoreactivity immunoreactivity (%) 

Sample No HG pTNM Tumour size ER IRS PR IRS Tumour ST Tumour ST 

1 II pT1N0 ≤2 cm 0 0 2 2 5 50 

2 III pT2N0 2-5 cm 8 6 2 2 5 50 

3 II pT1N0 ≤2 cm 9 0 2 Missing sample 90 Missing sample 

4 III pT1bN0 0,5-1 cm 1 0 0 0 0 0 

5 II pT2N0 2-5 cm 12 3 2 1 30 80 

6 II pT(m)3N0 >5 cm 4 9 0 0 0 0 

7 II pT2N0 2-5 cm 2 8 1 0 15 0 

8 II pT1(3)N0 ≤2 cm 3 6 0 0 0 0 

9 III pT1bN0 0,5-1 cm 0 0 0 0 0 0 

10 II pT1pN0 ≤2 cm 12 12 0 0 0 0 

11 II pT1cN0 1-2 cm 12 6 2 1 60 10 

12 III pT2N0 2-5 cm 8 2 1 1 25 40 

13 III pT2N0 2-5 cm 0 0 2 2 30 30 

14 III pT2N0 2-5 cm 0 0 2 2 30 30 

15 II pT2N0 2-5 cm 8 12 2 2 80 80 

16 III pT2N0 2-5 cm 0 0 2 2 80 80 

17 II p(m)T4dN0 

Inflammatory 

cancer 

12 12 1 1 30 80 

18 III pT2N0 2-5 cm 0 0 1 1 15 40 

19 II pT1cN0 1-2 cm 12 9 1 0 20 0 

20 I pT1N0

112 



The distribution of the variables was determined 

using distribution histograms with probability 

plots. The results were evaluated with nonparametric 

Mann-Whitney U test and with a two-tailed 

Spearman correlation test considering value 

p≤0,05 as statistically significant. Statistical 

analyses were performed in SPSS software, version 

17.0. 

RESULTS 

Tumour size and histological grade were determined 

in 50 primary invasive ductal breast cancer 

patients (Table 1, Table 2). Lymph node metastases 

were found in 26 primary invasive ductal 

breast cancer patients (52%). Estrogens and progesterone 

receptor status were also determined in 

patients with ductal invasive breast cancer (Table 

1, Table 2). In the lymph node-negative patients, 

18 tumours (75%) were positive and six tumours 

(25%) were negative to estrogens receptors, 16 

tumours (66,7%) were positive and eight tumours 

(33,3%) were negative to progesterone receptors 

(Table 1). In the lymph node-positive patients, 20 

tumours (76,9%) were positive and six tumours 

(23,1%) were negative to estrogens receptors, 17 

tumours (65,4%) were positive and nine tumours 

(34,6%) were negative to progesterone receptors 

(Table 2). 

Statistically significant differences between 

lymph node-negative and lymph node-positive 

breast cancer patients were noticed considering 

tumour size (p=0,041), but no significant differences 

were noticed considering histological grade. 

No significant differences between ER-IRS 

and PR-IRS were noticed considering the lymph 

node status. ER-IRS significantly correlated to 

PR-IRS regardless of the lymph node status (in 

patients with no metastasis p=0,000, in patients 

with metastasis p=0,001, respectively). In addition, 

statistically significant inverse correlation 

was found between ER-IRS and PR-IRS and histological 

grade in lymph node-negative patients 

(p=0,017, p=0,002, respectively). 

IL-18 was present in breast cancer tumour, only 

in the tumour cell cytoplasm (Figure 1), in the 

surrounding unchanged tissue of the same pati- 

Table 2. Pathohistological factors, intensity and extent of IL-18 immunoreactivity in patients with lymph node metastasis* 



immunoreactivity immunoreactivity (%) 

Sample No HG pTNM Tumour size ER IRS PR IRS Tumour ST Tumour ST 

1 II pT2N1 2-5 cm 8 1 2 2 90 90 

2 III pmT2(2)N3 2-5 cm 9 6 1 1 25 30 

3 II pT2N1 2-5 cm 0 0 2 2 10 25 

4 III pT2N1a 2-5 cm 0 0 2 Missing sample 5 Missing sample 

5 II pT1N1

ents and in breast tissue of patients with benign 

breast tumour or other benign breast disease. The 

extent of the expression of IL-18 significantly 

correlated to the intensity of the expression of IL- 

18 (p=0,000). Both the extent and the intensity of 

the expression of IL-18 were significantly higher 

in breast cancer tumour tissue compared to the 

surrounding unchanged tissue, with the exception 

for IL-18 immunoreactivity in the lymph nodenegative 

patients (Table 1, Table 2). In the lymph 

node-negative breast cancer patients, six tumours 

(25%) were negative to IL-18 and 18 tumours 

(75%) were positive from which one tumour 

(4,2%) was strongly positively elevated as score 

3 (Table 1). In the lymph node-positive patients, 

four tumours (15,3%) were negative to IL-18 and 

22 tumours (84,7%) were positive from which 

two tumours (7,7%) were strongly positively elevated 

as score 3 (Table 2). In patients with benign 

breast tumour or other benign breast diseases, 

seven breast tissue samples (35%) were negative 

to IL-18 and 13 breast tissue samples (65%) were 

positive from which none of them was strongly 

positively elevated as 3 (Table 3). There were 

no significant differences in the IL-18 expression 

between breast cancer tumour tissue and breast 

tissue of patients with benign breast tumour or 

other benign breast diseases (p=0,057) (Table 3). 

In addition, there were no significant differences 

in the IL-18 expression between lymph node-negative 

and the lymph node-positive breast cancer 

Table 3. The IL-18 immunoreactivity in patients with benign 

breast disease 

Sample No. 


immunoreactivity 


immunoreactivity (%) 

1 0 0 

2 0 0 

3 1 30 

4 2 80 

5 0 0 

6 1 60 

7 1 30 

8 1 60 

9 0 0 

10 1 90 

11 1 60 

12 0 0 

13 0 90 

14 1 60 

15 1 0 

16 2 80 

17 2 80 

18 2 80 

19 2 80 

20 0 0 

p=0,057* p=0,828* 

*p value (Mann-Whitney U test) for differences between breast 

cancer tumour tissue and breast tissue in patients with benign breast 

disease 


patients. Furthermore, no significant correlations 

of IL-18 expression to pathohistological factors 

(tumour size and histolgical grade) were found, 

as well as any significant correlation of IL-18 

expression to estrogens and progesterone receptor 

status. 

DISCUSSION 

The results of this study have shown that IL-18 

was present in breast cancer tumour tissue, in the 

surrounding unchanged tissue of the same patients, 

and in the breast tissue of patients with benign 

breast tumour or other benign breast disease. 

The IL-18 expression was significantly higher in 

tumour tissue compared to the surrounding unchanged 

breast tissue (Table 1, Table 2). 

There is evidence that some cytokines are produced 

by host stromal cells and immune cells, in 

response to molecules secreted by cancer cells or 

by cancer cells themselves (16). Therefore, neoplastic 

cells produce cytokines and chemokines 

that are mitogenic and/or chemoattractants for 

granulocytes, mast cells, monocytes/macrophages, 

fibroblasts and endothelial cells. In addition, 

activated fibroblasts and infiltrating inflammatory 

cells secrete proteolytic enzymes, cytokines 

and chemokines, which are mitogenic for neoplastic 

cells, as well as for endothelial cells involved 

in neoangiogenesis and lymphangiogenesis (3). 

These factors may potentiate tumour growth, stimulate 

angiogenesis, induce fibroblast migration 

and maturation, and enable metastatic spread via 

engagement of venous or lymphatic networks 

(3). It is possible that IL-18, which was highly 

expressed in tumour by comparison with surrounding 

unchanged breast tissue, is also being se- 

A B 

Figure 1. Microscopic view of IL-18 positive breast cancer 

tumour: A) IL-18 +1 positive tumour; B) IL-18 +3 positive 

tumour (Srabović Nahida, 2009) 

113

114 


creted by breast cancer cells. High serum levels 

of IL-18 were already reported in breast cancer 

patients (10-12). Presence of IL-18 in unchanged 

surrounding tissue in breast cancer patients 

and in breast tissue of patients with benign breast 

tumour or other benign breast diseases suggests 

that this cytokine may be produced in response 

to the surgical stress (17, 18), and/or as a part of 

inflammation process that often accompanies tumour 

growth (16). 

The results obtained in this investigation revealed 

no differences in IL-18 immunoreactivity between 

lymph node positive and lymph node negative breast 

cancer patients, as it was in some way expected. 

Furthermore, no significant correlations of IL- 

18 expression to pathohistological factors (tumour 

size, histological grade) were found. 

Recent clinical studies reported that serum IL-18 

levels were significantly increased in breast cancer 

patients when compared with controls (19) 

and significantly increased in patients with metastatic 

spread compared with those without it (12). 

Also it was found that serum IL-18 levels were 

significantly higher in patients whose tumour size 

was 5 cm or greater when compared to patients 

whose tumour size was 2 cm or less (19). These 

findings and results obtained in our study suggest 

that IL-18 may be useful as a prognostic marker in 

monitoring breast cancer patients and in tumour 

surveillance to detect distant metastases. 

Although no significant difference was observed 

in the IL-18 expression between the lymph node 

positive and the lymph node negative breast cancer 

patients, the overexpression of IL-18 with 

regard to the surrounding tissue in these patients 

may, alone, indicate possible involvement of this 

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2. 

3. 

4. 

Rao VSR, Dyer CE, Jameel JKA, Drew PJ, Greenman 

J. Potential prognostic and therapeutic roles for 

cytokines in breast cancer. Oncol Rep 2006; 15:179- 

85. 

Wilson J, Balkwill F. The role of cytokines in the 

epithelial cancer microenvironment. Semin Cancer 

Biol 2002; 12:113-20. 

Coussens LM, Werb Z. Inflammation and cancer. 

Nature 2002; 420: 860-7 

Lebel-Binay S, Berger A, Zinzindohoue F, Cugnenc 

P, Thiounn N, Fridman WH, Pages F. Interleukin-18: 

Biological properties and clinical implications. Eur 

Cytokine Netw 2000; 11:15-26 

cytokine in the complex pathogenesis of breast 

cancer, as previous clinical studies suggest. 

Results in this study have shown that IL-18 was 

present in breast cancer tumour cells only in the 

tumour cell cytoplasm (Figure 1). According to 

these findings it can not be certain that cancer 

cells produce this cytokine. Detailed tumour cell 

morphologic analysis can answer the question if 

cancer cell produce IL-18 and if this cytokine is 

in secretory pathway through Golgi apparatus. 

Furthermore, IL-18 serum level determinations, 

and tumour cells morphologic analysis, when 

combined may provide more detailed information 

about immunomodulating role of this cytokine in 

solid tumour tissue. That is an important limitation 

to our study because it is not possible to precisely 

evaluate exact role of IL-18 in the immunomodulating 

process in the breast carcinogenesis, 

and measurements of IL-18 serum concentrations 

together with tumour cells histological analyses 

are needed. Nevertheless, the results obtained in 

this study may, at least partly, contribute to better 

understanding of the complex breast cancer etiology, 

and may contribute to the development of 

the new diagnostic and therapeutic approaches to 

this severe disease. 



by Srabović N, Mujagić Z, Mujanović-Mustedanagić 

J, Muminović Z, Softić A, Begić L. 

The interleukins IL-13 and IL-18 in the primary 

breast cancer tumor tissue. Proceedings of the 

35th FEBS Congress Molecules of Life, Gothenburg, 

Sweden, June 26 – July 1, 2010. Abstract 

A2.04. FEBS 2010; 277(suppl.1): 43-4. 


5. 

6. 

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S, Namba M, Tanimoto T, Torigoe K, Fujii M, Ikeda 

M, Fukuda S, Kurimoto M. Interferon-gamma-inducing 

factor enhances T helper 1 cytokine production 

by stimulated human T cells: Synergism with interleukin-12 

for interferon-gamma production. Eur J 

Immunol 1996; 26:1647-51. 

Hoshino T, Wiltrout RH, Young HA. IL-18 is a potent 

coinducer of IL-13 in NK and T cells: a new 

potential role for IL-18 in modulating the immune 

response. J Immunol 1999; 162:5070-7.

7. Coughlin CM, Salhany KE, Wysocka M, Aruga E, 

Kurzawa H, Chang AE, Hunter CA, Fox JC, Trinchjieri 

G, Lee WM. Interleukin -12 and interleukin- 

18 synergistically induce murine tumor regression 

which involves inhibition of angiogenesis. J Clin 

Invest 1998; 101:1441-52. 

8. Tanaka F, Hashimoto W, Okamura H, Robbins PD, 

Lotze MT, Tahara H. Rapid generation of potent and 

tumor-specific cytotoksic T lymphocytes by interleukin-18 

using dendritic cells and natural killer cells. 

Cancer Res 2000; 60:4838-44. 

9. Kawabata T, Lehikura T, Majima T. Preoperative 

serum IL-18 level as post operative prognostic marker 

in patients with gastric carcinoma. Cancer 2001; 

92:250-5. 

10. Merendino RA, Gangemi S, Ruello A, Bene A, Losi 

E, Lonbardo G, Purello-Dambrosio F. Serum level 

of IL-18 and sICAM1 in patients affected by breast 

cancer:preliminary considerations. Int J Biol Markers 

2001; 16 ( suppl 2):126-9. 

11. Soheir AL, Eissa MD, Samar A, Zaki MD, Shereen 

M, El-Maghraby MD, Dalia Y, Kadry MD. Importance 

of serum IL-18 and RANTES as markers for 

breast carcinoma progression. J Egyptian Natl Canc 

Inst 2005; 17 (suppl 1):51-5. 

12. Gunel N, Coskun U, Sancak B, Gunel U, Hasdemir 

D. Clinical importance of serum IL-18 and nitric 

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(suppl 3):663-7. 

Ekspresija interleukina 18 u tkivu primarnog tumora raka dojke 

Nahida Srabović 1 , Zlata Mujagić 1 , Jasminka Mujanović-Mustedanagić 2 , Zdeno Muminović 2 , Elmir 

Čičkušić 2 

1 Katedra za biohemiju, Farmaceutski fakultet, Univerzitet u Tuzli; 2 Poliklinika za laboratorijsku dijagnostiku, Univerzitetsko klinički centar 

Tuzla; Tuzla, Bosna i Hercegovina 

SAŽETAK 


Cilj Utvrditi prisustvo i nivo ekspresije IL-18 u primarnom tumoru karcinoma dojke u odnosu na nepromijenjeno 

tkivo dojke iste bolesnice i na tkivo dojke bolesnica s benignim oboljenjem dojke. Ispitati 

korelaciju između nivoa ekspresije IL-18 i patohistoloških faktora, kao i korelaciju nivoa ekspresije 

IL-18 i statusa estrogenskih i progesteronskih receptora. 

Materijal i metode Prospektivna randomizirana studija provedena je na Poliklinici za laboratorijsku 

dijagnostiku Univerzitetskog kliničkog centra Tuzla i obuhvatila je 50 bolesnica s invazivnim duktalnim 

karcinomom dojke i 20 bolesnica s benignim tumorom dojke i drugim benignim oboljenjima dojke. 

Trostepena imunohistohemijska metoda korištena je za određivanje nivoa ekspresije IL-18, kao i statusa 

hormonskih receptora. 

Rezultati IL-18 bio je prisutan i u tumoru karcinoma dojke i u okolnom nepromijenjenom tkivu, kao i u 

tkivu dojke bolesnica s benignim oboljenjima dojke. Ekspresija IL-18 u tumoru karcinoma dojke bila je 

značajno veća u odnosu na okolno tkivo (p

116 


Use of amino-terminal pro-B type natriuretic peptide as the parameter 

for long term monitoring of water overload in patient 

with chronic kidney diseases 

Besim Prnjavorac 1,2 , Kadrija Abduzaimović 1 , Jasna Jukić 1 , Rifat Sejdinović 1 , Ekrema Mujarić 3 , Nedžada 

Irejiz 1 , Almira Hadžović-Džuvo 4 , Radivoj Jadrić 4 , Adlija Čaušević 5 , Sabina Semiz 5 , Tamer Bego 5 , Maja 

Malenica 5 

1 2 Department of Internal Medicine, General Hospital Tešanj, Tešanj, Department of Patophysiology, Shcool of Pharmacy, University of 

Sarajevo, Sarajevo, 3Department of Internal Medicine, Cantonal Hospital Zenica, Zenica, 4Department of Physiology and Biochemistry, 

University of Sarajevo, Sarajevo, 5Department of Physiology and Biochemistry, School of Medicine, University of Sarajevo, Sarajevo; 


Corresponding autor: 

Besim Prnjavorac 

Department of Internal Medicine, 

General Hospital Tešanj 

Braće Pobrić 17, 74260 Tešanj, 


Phone: +387 32 656 300 

E-mail: pbesim@bih.net.ba 





Accepted: 


ABSTRACT 


Aim To analyze usefulness of measurement amino-terminal pro-B 

type natriuretic peptide of (NT pro-BNP) as the one of parameters 

of water overload in patients with chronic kidney diseases. 

Mmethods A total number of 277 patients with chronic kidney diseases 

(CKD) were followed up in the period of ten years between 

January 2000 and July 2010. Patients with creatinine clearance 

of 60 ml/min or less were included in the study. Changes of creatinine 

clearance, and in last five years changes of NT pro-BNP 

were followed. Water overload was analyzed using chest x-ray in 

relation with concentration of NT pro-BNP in the blood. 

Results Decrease of clearance of creatinine ranged from average 

54,7 ml/min in the first year to 14,6 ml/min in the fifth year of 

the monitoring. Average NT pro-BNP level in patients without any 

sign of water overload was 94 pg/ml (SD 21), mean value in those 

with Kerley lines was 231 pg/ml/L (SD 64), in those with clear 

signs of water overload but without pleural effusion it was 525 

pg/ml (SD 223), and in those with water retention including pleural 

effusion it was 1606 pg/ml (SD 1134). Using test of multiple 

correlation a statistically significant correlation between X-ray signs 

of water overload and NT pro-BNP concentration was shown, 

p

INTRODUCTION: 

Water overload is one of the common reasons to 

begin chronic dialysis treatment. Hyperhydration 

induces retention of fluid in mesenhimal tissue 

long time before any patient’s symptom or chest 

X-ray signs. If any degree of water overload is 

present, near all tissue in the body are in harm. 

So, observance of water overload is very important 

in any stage of chronic kidney disease (1). 

The B-type, or brain natriuretic peptide (BNP) is 

a 32 amino acid peptide hormone predominantly 

released from the ventricules in response to left 

ventricul (LV) volume expansion and pressure 

overload, and has a circulating half-life of about 

30 minutes. N-terminal pro-BNP (NT-pro-BNP) 

is a 76 amino acid peptide hormone, also cleaved 

from the prohormone; it is biologically inactive 

but has a much longer half-life (about 120 minutes) 

(2,3). NT pro-BNP levels are well known 

to correlate with left ventricul filling pressures 

and are elevated in patients with systolic heart 

dysfunction (4,5). Both BNP and their precursor 

NT-pro-BNP levels were increased in proportion 

to the severity of heart failure as assessed using 

the New York Heart Association classification in 

the general population (6,7). 

Analysis of usefulness of measurement of NT 

pro-BNP, as laboratory parameter of water overload, 

is the aim of this study. 


Patients with chronic kidney diseases were followed 

up during ten years, since January 2000 

to July 2010. Patients with creatinine clearance 

60 ml/min or less were included in this study. Laboratory 

examination was prepared every three 

months, or sooner, according to general clinical 

status. Among others, changes of creatinine clearance 

and total body water (increase or not of 

body mass between two controls, if no signs of 

water overload on chest X-ray) were followed. 

Table 1. Parameter measured during follow up* 

During the last five years measurement of N-terminal 

pro-BNP, which is precursor of atrial natriuretic 

peptide was performed. Estimate of water overload 

was performed by X-ray examination. 3 degrees 

of X-ray signs of water overload were estimated 

(I-degree Kerley lines only, II-degree large signs of 

water retention but without pleural effusion, III-degree 

lung stasis with pleural effusion on X-ray of the 

chest). For statistical purposes analyses were performed 

for changes in clearance of creatinine, uric 

acid, NT pro-BNP and results of X-ray examination 

of the chest, according to the estimated degree. NT 

pro-BNP was measured using Roche CARDIAC 

cobas reader with enzyme-immunoassay method. 

ANOVA test of variance and test of multiple correlations 

have been performed. 

RESULTS 

During the last ten years we followed up all 277 

patients, 141 of them being female and 136 male 

patients, of average age 54,8 year (SD 6,4), ranging 

from 27 to 84. The mean value of the clearance 

of creatinine in the first year of monitoring 

was 54,7 ml/min (SD 8,6), in the second year 

44,8 ml/min (SD 8,1), in the third year 37,2 ml/ 

min (SD 6,4), in fourth year 19,2 ml/min (SD 

4,8), and in the fifth year 14,6 ml/min (SD 4,3). 

(Table 1) 153 out of all patients had increased 

uric acid. BNP concentration varied from 60 pg/ 

ml to 14480 pg/ml. The average NT pro-BNP level 

in patients without any signs of water overload 

on X-ray of the chest was 94 pg/ml (SD 21), 

mean value in those with Kerley lines was 231 

pg/ml (SD 64), in those with clear signs of water 

retention, but without pleural effusion it was 525 

pg/ml (SD 223), and in those with water retention 

including pleural effusion it was 1606 pg/ml (SD 

1134). (Figure 1, Figure 2) 

In the patients without water retention average 

serum concentration of uric acid was 386 µmol/L 

(SD 38), in those with different degree of water 

overload, described above, the mean values of 

Year of follow up 1st (2006) 2nd (2007) 3rd (2008) 4th (2009) 5th (2010) 

Creatinin level in µmol/L (SD) 147(76) 191(82) 254 (117) 337 (125) 461(134) 

Creatinine clearance in ml/min (SD) 54,5(8,6) 44,8(8,1) 37,2(6,4) 19,2 (4,8) 14,6 (4,3) 

Uric acid in µmol/L (SD 397 (45) 425 (61) 448 (63) 468 (78) 516 (82) 

NT proBNP pg/ml (No changes in chest X-ray) 83 (39) 91 (44) 96 (59) 102 (66) 98 (86) 

NT proBNP pg/ml (Kerley lines only in chest X-ray) 149 (47) 188 (56) 233 (75) 246 (67) 342 (73) 

NT proBNP pg/ml – signes of stasis in chest X-ray) 287 (119) 366 (172) 588 (212) 627 358) 761 (284) 

NT proBNP pg/ml - signes of stasis and pleural effusion in chest X-ray) 726 (449) 1184 (773) 2187 (1316) 1689 (988) 2248 (725) 

*annual mean values and standard deviation (SD) were shown; NT pro-BNP given in pg/ml 

Prnjavorac et al NT pro-BNP in chronic kidney disease 

117

118 


Figure 1. chest X-ray with pleural effussion as a sign of water 

overload (Prnjavorac B., 2010) 

uric acid were 426 µmol/L (SD 76), 489 µmol/L 

(SD 86), and 547 µmol/L (SD 84), respectively. 

Using test of multiple correlation we have shown 

a statistically significant correlation between Xray 

signs of water overload and NT pro-BNP concentration 

on the level of p



by Prnjavorac B, Jukić J, Abduzaimović K, 

Mujarić E. Analysis of laboratory parameters in 

estimation of overall water balance of the body. 

Proceedings of the 1st Congress of Medical Bi- 

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20. Spanaus KS, Kronberg F, Ritz E, Fliser D, Hersberger 

M, Kollerits B, Konig P, von Eckardstein A. Btype 

of natriuretic peptide concentration predict the 

progression of nondiabetic chronic kidney disease: 

Mild-to-moderate kidney disease study. Clin Chem 

2007; 53: 264-72. 

21. De fillipi C, van Kimmeande RR, Pinto YM. Amino-terminal 

pro-B-type natriuretic peptide testing in 

renal disease. Am J Cardiol 2008; 101:82-8. 

22. Srisawasdi P, Vanavanan S, Charoenpanichkit C, 

Kroll hM. The effect of renal dysfunction on BNP , 

NT-proBNP, and their ratio. Am J Clin Pathol 2010; 

133:14-23. 

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Analiza amino-terminalnog pro-B-tipa natriuretskog peptida kao parametra retencije 

tekućine u praćenju bolesnika s hroničnom bubrežnom bolesti 

Besim Prnjavorac1,2 , Kadrija Abduzaimović1 , Jasna Jukić1 , Rifat Sejdinović1 , Ekrema Mujarić3 , Nedžada 

Irejiz1 , Almira Hadžović-Džuvo4 , Radivoj Jadrić4 , Adlija Čaušević5 , Sabina Semiz 5 , Tamer Bego5 , Maja 

Malenica5 1 2 Odjeljenje interne medicine, Opća bolnica Tešanj, Tešanj; Katedra za patološku fiziologiju, Farmaceutski fakultet Univerziteta u Sarajevu, 

Sarajevo; 3Odjeljenje interne medicine, Kantonalna bolnica Zenica, Zenica; 4Institut za fiziologiju i biohemiju, Medicinski fakultet 

Univerziteta u Sarajevu, Sarajevo; 5Katedra za biohemiju, Farmaceutski fakultet Univerziteta u Sarajevu, Sarajevo; Bosna i Hercegovina 

SAŽETAK 

Cilj Analizirati mogućnost upotrebe mjerenja plazmatske koncentracije NT pro-BNP, kao prekursora 

aktivnog atrijskog natriuretskog peptide, u ocjeni retencije tekućine kod pacijenata s hroničnim bolestima 

bubrega. 

Metode Rad predstavlja analizu desetogodišnjeg praćenja 277 pacijenata s hroničnim bolestima bubrega, 

s klirensom kreatinina 60 ml/min ili manje. Analizirane su promjene klirensa kreatinina tokom 

godina praćenja, a posljednjih pet godina praćene su i vrijednosti NT pro-BNP. Stanje opće hidracije 

organizma pacijenata, te retencija vode, praćeni su analizom rtg snimka pluća, a u posljednjih pet godina 

i praćenjem promjena NT pro-BNP, te usporedbom promjene njegove koncentracije i promjena 

na rendgenogramu pluća. Od ostalih praćenih parametara funkcije bubrega, u ovom su radu analizirani 

nivoi kreatinina i mokraćne kiseline. 

Rezultati Registriran je očekivani pad klirensa kreatinina, od prosječno 54,7 ml/min (SD 6,4) u prvoj 

godini praćenja, do prosječno 14,6 ml/min (SD 4,3) u petoj godini praćenja. NT pro-BNP bio je u približno 

normalnim vrijednostima, prosječno 94 pg/ml (SD 21) kod pacijenata bez uočljivih zastojnih 

promjena na rentgenogramu pluća. Kod pacijenata s prisutnim Kerleyevim linijama, prosječna razina 

NT pro-BNP bila je 231 pg/ml (SD 64); kod pacijenata s veoma izraženim zastojnim promjenama, ali 

bez pleuralnog izljeva, bila je 525 pg/ml (SD 223); dok je prosječan nivo NT pro-BNP kod pacijenata 

s izljevom bio 1606 pg/ml (SD 1134). Testom multiple korelacije nađena je statistička povezanost progresije 

promjena na rentgenogramu pluća i nivoa NT pro-BNP na razini p


B-type natriuretic peptide (BNP) serum levels in rats after forced 

repeated swimming stress 

Almira Hadžovic-Džuvo 1 , Amina Valjevac 1 , Nesina Avdagić 1 , Orhan Lepara 1 , Asija Zaćiragić 1 , Radivoj 

Jadrić 2 , Jasmin Alajbegović 3 , Besim Prnjavorac 4 

1 Department of Physiology, 2 Department of Biochemistry, 3 School of Medicine, 4 School of Pharmacy; University of Sarajevo, Bosnia 

and Herzegovina 


Almira Hadžović-Džuvo 

School of Medicine, 




Phone: +387 33 651 014; 

Fax.: +387 33 663 743 

Email: fiziologija.09@gmail.com 





Accepted: 


ABSTRACT 


Aim To estimate the effects of forced repeated swimming stress on 

BNP serum levels in rats. 

Methods Adult male Wistar rats weighting between 280-330 

g were divided into two groups: control group (n =8) and stress 

group (n =8). Rats in the stress group were exposed to forced 

swimming stress daily, for 7 days. The rats were forced to swim 

in plastic tanks (90 cm wide, 120 cm deep) containing tap water 

(temperature ca. 25˚C). The depth of water was 40 cm. Duration of 

each swimming session progressively increased from 10 minutes 

on the first day to 40 minutes on days 6 and 7. Rats were sacrificed 

and blood was drawn from abdominal aorta for BNP analysis 

immediately after the last swimming session. B-type natriuretic 

serum level was determined by ELISA method using RAT BNP-32 

kit (Phoenix Pharmaceutical Inc.). 

Results There was no statistically significant difference between 

mean BNP serum level in the stress group after the swimming period 

(0.81±0.14 ng/ml) as compared to the unstressed group of rats 

(0.8 ±0.08ng/ml). After the swimming period mean body weight 

slightly decreased in the stress group in comparison with values 

before stress period (296.3 g vs.272.8 g), but this difference was 

not statistically significant. The stress period had no influence on 

food intake in the stress rat group. 

Conclusion The workload consisting of 40-minutes long swimming 

session is not sufficient to provoke BNP release from myocardium 

in rats. 

Key words: BNP, rats, swimming, stress 

121

122 


INTRODUCTION 

B-type natriuretic peptide (BNP) is a hormone 

which is secreted from the ventricle in response 

to elevated volume and filling pressures. Recent 

studies showed that BNP has an impact on the 

regulation of hypothalamic-pituitary–adrenocortical 

(HPA) responses to physiological and psychological 

stressors (1). 

An exercise, as a physiological stressor, has an 

impact on BNP serum levels, but most of the 

studies were conducted on patients with altered 

myocardial functions. Those studies showed 

elevation of BNP levels including an inactive 

form of BNP prohormone (NTproBNP) immediately 

after the exercise stress testing, but only 

in subjects with associated myocardial ischemia 

(2,3). These results confirmed an important link 

between the severity of an acute ischemic insult 

and the circulating levels of BNP. At the same 

time, recent findings showed benefit of regular 

exercise on BNP levels in patients with heart failure 

(4). 

Results of studies assessing the impact of acute 

exercise on BNP levels in healthy humans are 

insufficient and controversial. Elevated natriuretic 

peptide levels were detected in healthy endurance 

male athletes immediately after prolonged 

strenuous exercise and interpreted as the result 

of exercise-induced sub-clinical myocardial cell 

damage (5). Conversely, in female marathon 

runners elevated BNP levels were found only one 

day after the marathon race (6). A recent study 

also showed that short-term maximal physical 

exercise in healthy individuals led to a fast and 

transient rise of plasma BNP (7). 

For the proper interpretation of BNP levels during 

the stress testing, there is a need for further 

studies on BNP hemodynamic changes during 

the exercise. Swimming in small laboratory 

animals has been widely used for studying the 

physiological changes and the organism’s capacity 

to respond to the stress. Increased myocardium 

wall stress rapidly triggers synthesis and release 

of natriuretic peptides in rats (8), but there is no 

conclusive effect of swimming exercise on BNP 

levels in rats. 

Therefore, the aim of this study was to estimate 

the effects of forced repeated swimming stress on 

BNP serum levels in rats 

MATERIALS AND METHODS 

Adult male Wistar rats (weight 300-350 g) were 

divided into control group (n =8) and stress group 

(n =8). All the rats were given standard rat 

chow and tap water ad libitum and were housed 

at 25±3º C on a 12-hour dark/light cycle. 

All the experimental procedures were performed 

at the School of Medicine in Sarajevo, Department 

of Physiology and Biochemistry and 

approved by the Ethic Committee of the School 

of Medicine in Sarajevo. 

The stress group of rats was exposed to forced 

swimming stress daily, between 10.00 AM to 

11.00 AM, for 7 days. Duration of each swimming 

session progressively increased from 10 

minutes on the first day to 40 minutes on days 

6 and 7 in order to attain the adaptation of the 

rats. The rats were forced to swim in plastic 

tanks (90 cm wide, 120 cm deep), containing 

tap water (temperature of ≈25˚C). The depth of 

water was 40 cm. A maximum of two rats were 

allowed to swim together during the stress session. 

The control rats were housed under the 

same conditions and they were exposed to same 

procedure as the stress group. The body weight 

of each rat was measured before and after 

the stress period. 

Immediately after the last swimming session rats 

were anaesthetized, sacrificed and blood was 

drawn from abdominal aorta. After 30 minutes 

whole blood was centrifuged and the obtained 

serum was immediately frozen and stored at 

–20°C. 

Serum BNP levels were determined by ELI- 

SA method using RAT BNP-32 kit (Phoenix. 

Ph.Coperation) according to the manufacturer’s 

instructions. 

Data are presented as mean ± standard error of 

the mean (SEM). Analysis was performed using 

SPSS package 16.0 (SPSS Inc., Chicago, Illionis, 

USA). Changes in parameters before and after 

the swimming stress period were compared using 

non-parametric equivalent to paired t-tests, Wilcoxon 

test. Differences in mean values between 

groups were assessed using the Mann-Whitney 

test. A two-tailed p value< 0,05 was considered 

significant.

RESULTS 

There were no statistically significant differences 

in body weight (291,4±13,7 v. 296,3±14,1) 

between control and swimming group at the 

beginning of the experiment (before the stress 

period) (Table 1). Swimming stress slightly decreased 

mean body weight in the stress group in 

comparison with values before the stress period 

(296.3 g vs.272.8 g) but this difference was not 

statistically significant (Table 1). 

The mean daily food intake was 21,7 ± 2,3 grams 

per rat in the control group. In the stress group 

daily food intake measured at the beginning of 

the experiment was higher (22,4±1,4 grams/rat/ 

day) in comparison with values in the control 

group, but this difference was not statistically 

significant. The stress period had no influence 

on food intake in the stress rat group (21,4±2,3 

grams/rat/day) (Table 1). 

Mean serum BNP level in the stress group after 

the swimming period (0.81±0.14 ng/ml) was not 

significantly different as compared to BNP level 

in unstressed group of rats (0.8 ±0.08ng/ml) (Figure 

1). 

DISCUSSION 

The main stimulus for BNP secretion from ventricles 

is the increased wall stress induced by 

volume and pressure overload (9). Exercise, as 

a physiological stressor, elevates sympathetic 

activity leading to the increase in heart rate and 

myocardial contractility. That could be a possible 

stimulus for BNP ventricle secretion (7). But results 

of previous studies on BNP response during 

exercise are inconsistent (5-7). 

Most studies performed on human subjects with 

altered myocardial function showed an increase 

of BNP levels during exercise testing, especially 

in patients with heart failure and acute coronary 

Table 1. Mean values of body weight, food intake and BNP 

concentration in rats before and after the physiological stress 

(swimming) and in the control group of rats* 

Parameters 

Control 

group (n=8) 

Stress (swimming) group 

(n=8) 

Before stress 

period 

After stress 

period 

Body weight (g) 291,4±13,7 296,3±14,1 272,8±21,4 

Food intake (g/ 

day/rat) 

21,7 ± 2,3 

22,4±1,4 

(1. day) 

21,4±2,3 

(7.day) 

BNP (pg/ml) 0,80±0,02 0,81±0,06 

*results are presented as mean values ± SEM 

Hadžović-Džuvo et al Effect of swimming on BNP serum levels in rats 

syndrome (3). Previous studies also showed that 

BNP could have a possible role in the cardiac 

adaptation to acute exercise (10). Elevations of 

cardiac biomarkers can be present in elite and in 

recreational athletes, especially after prolonged 

and strenuous endurance exercise bouts (e.g., 

marathon and ultra triathlon) (5, 6). However, authors 

of those studies concluded that in contrast 

to cardiac patients, it is still unclear if the exercise-associated 

alteration or increase in cardiac biomarkers 

in obviously healthy athletes represents 

clinically significant cardiac insult or whether it 

is indeed a part of the physiological response to 

endurance exercise (11). 

Our results showed that 40-minute forced swimming 

stress did not cause the increase in BNP 

serum concentration in rats. We were not able to 

compare our study with any previous studies on 

BNP response during the swimming stress in rats. 

Our results are not in concordance with results 

of the study performed by Magga et al. where 

the aim was to evaluate the mechanisms of brain 

natriuretic peptide (BNP) gene expression in 

response to acute cardiac overload (from 30 min 

to 4 h) after arginin-vasopressin injection, in normal 

and hypertrophied rats myocardium (8). The 

results from the above mentioned study showed 

that pressure overload rapidly stimulates BNP 

gene expression in hearts of normal and hypertensive 

rats. Natriuretic peptide gene expression 

followed by increased plasma BNP levels started 

within 1 hour and peak BNP values were detected 

at 4 hours after. This could be explained by 

Figure 1. B-type natriuretic peptid levels in control and stress 

group* 

results are presented as mean values ± SEM (not significantly 

different) 

123

124 


the fact that BNP response depends on a stimulus 

that exerts pressure overload. Swimming or 

other exercise stimuli, as physiological stressors, 

provoke numerous endocrine responses that could 

influence BNP secretion. It is possible that 

BNP secretion during the exercise could be a part 

of exercise-induced hormonal response but this 

response needs an adequate stimulus. This fact 

is supported by findings of many studies on healthy 

population that are in concordance with our 

results. Novel data of Wozakovska-Kapłon et al. 

(12) showed an increase of BNP plasma levels 

after treadmill ergometry in patient with atrial fibrillation, 

but not in the control healthy group. 

An important fact is also that most of the studies 

on healthy population included athletes from endurance 

discipline like marathon, ultra marathon 

etc. (5,6). Taking into consideration our results, 

and the fact that 40- minute swimming exerts significantly 

lower workload as compared to overload 

during endurance sports, we assume that 

BNP secretion during the exercise is not only a 

response to the increased wall stress, but represents 

a more complex response which possibly 

has a protective role in cardiomyocites due to a 

stressful situation. Therefore, locally generated 

BNP in the heart muscle may play a significant 

role in cardiac adaptation to acute changes in 

mechanical load. We assume that this protective 

response has to be proceeded with adequate 

workload which probably also leads to some ultra 

alterations of myocardial structures. Study by 

Scharhag et al. (10) showed exercise-associated 

alterations of cardiac biomarkers troponin T and 

I as well as ischemia-modified albumin, which 

also supports this view. 

Many discrepancies in the results on BNP dynamic 

during the exercise in different studies allow 

us to suggest different possible causes of the 

observed results. Considering the study by Krupicka 

et al. (7) which confirmed treadmill exerci- 

REFERENCES 

1. Amir O, Sagiv M, Eynon N, Yamin C, Rogowski O, 

Gerzy Y, Amir RE. The response of circulating brain 

natriuretic peptide to academic stress in college students. 

Stress 2010; 13:83-90. 

2. Sabatine MS, 

Morrow DA, de Lemos JA, Omland 

T, Desai MY, Tanasijevic M, Hall C, McCabe CH, 

Braunwald E. Acute changes in circulating natriuretic 

peptide levels in relation to myocardial ischemia. 


se in humans leads to modest and transient serum 

BNP elevation, which returns to baseline values 

in 10-15 minutes after the exercise, our results 

could also be explained by the fact that the period 

between the end of the swimming exercise 

and subsequent blood withdrawal, that is required 

by the protocols, is too long for the assessment 

of BNP changes as a response to this form 

of physiological stress. We propose modification 

in rats exercise protocols for assessment of BNP 

hemodynamic changes. 

In conclusion, the workload consisting of 40-minute 

long swimming session is not sufficient to 

provoke BNP release from myocardium in rats. 

Blood sampling from the rats should be performed 

as soon as possible, preferably from the tail 

vein, immediately after the stress exercise and 

workload in order to study myocardial responses 

to physiological stressors. 


This study was partly presented as an oral presentation 

by Hadžović-Džuvo A, Valjevac A, Avdagic 

N, Lepara O, Zaciragic A, Jadric R, Alajbegovic 

J. The effect of forced repeated swimming 

stress on B-type natriuretic peptide serum levels 

in rats. Proceedings of the 15th Annual Congress 

of the European College of Sports Sciences, Antalya, 

Turkey, June 23-26, 2010. OP-PH06 Physiology 

6 - Hormonal responses, Book of Abstracts 

of the 15th Annual Congress of the European 

College of Sport Science. European College of 

Sport Science, 2010, p. 235. 

Experimental work was financially supported by 

the Federal Ministry of Science and Education, 

Bosnia and Herzegovina, as a part of the project 

”Endothelin-1, N-terminal brain natriuretic peptide 

and adipocytokines serum levels in rats with 

streptozotocin induced diabetes mellitus’’ 


Sarullo F 

3. M, Gristina T, Brusca I, Serio G, Taormina 

A, La Chiusa SM, Castello A, Borruso E, Paterna 

S, Di Pasquale P. Usefulness of N-terminal pro-Btype 

natriuretic peptide levels in predicting residual 

myocardial ischemia in patients with ST elevation 

acute myocardial infarction. Minerva Cardioangiology 

2007; 55:149-55.

4. Smart NA, 

Steele M. Systematic review of the effect 

of aerobic and resistance exercise training on systemic 

brain natriuretic peptide (BNP) and N-terminal 

BNP expression in heart failure patients. Int J Card 

2010; 140:260-5. 

5. Scharhag J, Herrmann M, Urhausen A, Haschke M, 

Herrmann W, Kindermann W. Independent elevations 

of N-terminal pro-brain natriuretic peptide and 

cardiac troponins in endurance athletes after prolonged 

strenuous exercise. Am Heart J 2005; 150:1128- 

34. 

6. Frassl W, Kowoll R, Katz N, Speth M, Stangl A, 

7. 

Brechtel L, Joscht B, Boldt LH, Meier-Buttermilch 

R, Schlemmer M, Roecker L, Gunga HC.Cardiac 

markers (BNP, NT-pro-BNP, Troponin I, Troponin T, 

in female amateur runners before and up until three 

days after a marathon. Clin Lab 2008; 54:81-7. 

Krupicka J, Janota T, Kasalová Z, Hradec J. Ef- 

fect of short-term maximal exercise on BNP plasma 

levels in healthy individuals. Physiol Res 2010; 

59:625-8. 

8. Magga J, Marttila M, Mäntymaa P, Vuolteenaho 

O, Ruskoaho H. Brain natriuretic peptide in plasma, 

atria, and ventricles of vasopressin- and phenylephrine-infused 

conscious rats. Endocrin 1994; 

134:2505-15 

9. Palazzuoli A, Gallotta M, Quatrini I, Nuti R. Natriu- 

retic peptides (BNP and NT-proBNP): measurement 

and relevance in heart failure. Vasc Health Risk Manag 

2010; 6:411-8. 

10. Scharhag J, George K, Shave R, Urhausen A, Kin- 

dermann W. Exercise-associated increases in cardiac 

biomarkers. Med Sci Sports Exerc 2008; 40:1408- 

15. 

11. Nayanatara AK, Nagaraja HS, and Anupama BK. 

The effect of repeated swimming stress on organ 

weights and lipid peroxidation in rats. Thai J Physiol 

Sci 2005; 18:3-9. 

12. Wozakowska-Kaplon B, Opolski G. Exercise-in- 

duced natriuretic peptide secretion predicts cardioversion 

outcome in patients with persistent atrial 

fibrillation: discordant ANP and B-type natriuretic 

peptide response to exercise testing. Pacing Clin 

Electrophysiol 2010; 33:1203-9. 

Koncentracija B-tipa natriuretskog peptida (BNP) u serumu štakora nakon forsiranog, 

ponavljanog stresa, uzrokovanog plivanjem 

Almira Hadžovic-Džuvo1 , Amina Valjevac1 , Nesina Avdagić1 , Orhan Lepara1 , Asija Zaćiragić1 , Radivoj 

Jadrić2 , Jasmin Alajbegović3 , Besim Prnjavorac4 1 2 3 4 Katedra za fiziologiju, Katedra za biohemiju; Medicinski fakultet, Farmaceutski fakultet; Univerzitet u Sarajevu, Sarajevo, Bosna i 

Hercegovina 

SAŽETAK 

Hadžović-Džuvo et al Effect of swimming on BNP serum levels in rats 

Cilj Procijeniti efekte forsiranog, ponavljanog stresa, uzrokovanog plivanjem, na koncentraciju B-natriuretskog 

peptida u serumu štakora. 

Metode U studiji su korišteni odrasli mužjaci Wistar štakori, prosječne težine 280-330 g, koji su bili 

podijeljeni u dvije grupe, odnosno u kontrolnu (n=8) i stres grupu (n=8). Stres grupa štakora bila je 

izložena forsiranom, ponavljanom stresu, uzrokovanom svakodnevnim plivanjem, u trajanju od 7 dana. 

Sesija plivanja se progresivno povećavala, od 10 minuta prvog dana, pa do 40 minuta šestog i sedmog 

dana. Štakori su forsirani da plivaju u plastičnim bazenima (širine 90 cm i dubine 120 cm, prosječne 

temperature vode oko 25˚C). Dubina vode u bazenu iznosila je oko 40 cm. Odmah nakon posljednje sesije 

plivanja (sedmog dana) životinje su žrtvovane, a uzorak krvi za BNP analizu uzet je iz abdominalne 

aorte. Koncentracija BNP-a u serumu štakora određena je ELISA metodom, koristeći komercijalni kit 

RAT BNP-32 (Phoenix Pharmaceutical Inc.). 

Rezultati Nisu utvrđene statistički značajne razlike između srednjih vrijednosti koncentracije BNP-a u 

serumu stres grupe štakora, nakon perioda plivanja (0.81±0.14 ng/ml), u odnosu na vrijednosti utvrđene 

u kontrolnoj grupi štakora (0.8±0.08 ng/ml). Nakon stres perioda prosječne vrijednosti tjelesne mase 

štakora bile su neznatno niže u odnosu na vrijednosti tjelesne mase prije stres perioda (296.3 g vs. 272.8 

g), ali razlika nije bila statistički značajna. Stres period nije uticao na količinu konzumirane hrane u 

stres grupi štakora. 

Zaključak Radno opterećenje, izazvano 40-minutnom sesijom plivanja, nije dovoljno da izazove porast 

koncentracije BNP-a u serumu štakora. 

Original submission: 07 October 2010; Revised submission: 28 November 2010; Accepted: 04 December 2010. 

125

126 


Association of homocysteine with traditional and non-traditional 

risk factors in patients with atherosclerotic vascular disease 

Emina Kiseljaković 1 , Amina Valjevac 2 , Sabaheta Hasić 1 , Emina Nakaš-Ićindić 2 , Alen Džubur 3 , Radivoj 

Jadrić 1 

1 2 3 Department of Medical Biochemistry, Department of Human Physiology; Medical Faculty, University of Sarajevo, Clinic of Heart and 

Rheumatic Diseases, University of Sarajevo Clinical Centre; Bosnia and Herzegovina 


Emina Kiseljakovic 

Department of Medical Biochemistry, 

Medical Faculty, 


Čekaluša 90, 71 000 Sarajevo; 

Phone: +387 33 663 743; 

E-mail: eminacengic@hotmail.com 




2010; 

Accepted: 


ABSTRACT 


Aim To assess the association between total homocysteine (tHcy) 

and traditional and nontraditional risk factors in patients with atherosclerotic 

vascular disease (ASVD). 

Methods This cross-sectional study included 99 ASVD patients 

and 40 control subjects in whom we determined lipid profile, high 

sensitivity C-reactive protein (hsCRP), uric acid (UA) and tHcy. 

Results The median tHcy concentration was significantly higher 

in ASVD group compared to the controls ((18.7(13.65-24.45) vs. 

11.48(10.03-14.2) µmol/L (p

INTRODUCTION 

According to the American Heart Association 

(2002), atherosclerosis is a disease of large and 

medium-sized arteries characterized by thickening 

and hardening of the vascular wall. Atherosclerosis, 

also known as atherosclerotic vascular disease, 

or ASVD, is a chronic inflammatory disease with 

onset prior to its clinical manifestation (1). 

There are traditional, well established risk factors 

for atherosclerosis: age, hypertension, smoking, 

total cholesterol, high-density lipoprotein cholesterol 

(HDLc), low-density lipoprotein cholesterol 

(LDLc), triglycerides, glucose and physical inactivity 

(2). These traditional risk factors cannot 

fully explain changes on blood vessels so it is necessary 

to find “nonlipid” risk factors for ASVD 

(2). Almost 25% of patients with premature cardiovascular 

disease do not have any established 

risk factors (3). In addition to established cardiovascular 

risk factors, clinical research has identified 

more than 100 other conditions that may be 

associated with an increased risk for cardiovascular 

disease (4). Based on growing evidence, 

non-traditional risk factors for atherosclerosis 

have been identified: total homocysteine (tHcy) 

and uric acid (UA) as markers of endothelial dysfunction, 

C reactive protein (CRP) and fibrinogen 

as markers of inflammation and lipoprotein(a) as 

a marker of lipoprotein metabolism (5,6). 

Homocysteine is a sulfur-containing, non-protein 

amino acid that is formed during catabolism of 

essential amino acid methionine. This by-product 

of methyl-transfer reactions is important for the 

DNA synthesis, methylation of proteins, neurotransmitters 

and phospholipids (7). Homocysteine 

might be involved in initiation and progression 

of atherosclerosis through several mechanisms 

including disturbed lipid metabolism, increased 

production of reactive oxygen species, endothelial 

cellular damage, enhancement of LDL oxidation, 

inflammatory response, reduced activity of 

glutathione peroxidase, smooth muscle cell proliferation 

and promotion of endothelial-leukocyte 

interaction (8, 9). Stimulation of procoagulant 

and impairment of anticoagulation and fibrinolytic 

pathways caused by tHcy are responsible for 

atherothrombotic events (10). 

Elevated serum tHcy level, hyperhomocysteinemia 

(HHcy), have been associated with cardiovascular 

diseases (11,12). This standpoint mostly relies on 

Kiseljaković et al Homocysteine and risk factors 

results obtained from case-control studies which 

have consistently related serum tHcy elevation 

with myocardial infarction, stroke and atherothrombosis. 

Prospective observational studies, on 

the other hand, have been less convincing (13). 

It is the matter of debate whether the hyperhomocysteinemia 

is a cause or a marker of the atherosclerotic 

vascular disease, since it has been 

observed that homocysteine level increseas after 

vascular events (14, 15). Addressing the association 

between homocysteine and traditional 

and non-traditional risk factors might serve for 

better understanding of the pathophysiological 

mechanism of homocysteine induced atherosclerotic 

vascular disease. Significant associations 

exist between established and new risk factors, 

and better understanding of new risk factors may 

shed light on the pathogenetic mechanisms of established 

risk factors (16). 

The aim of the study was to assess the association 

between homocysteine and traditional and nontraditional 

risk factors in patients with atherosclerotic 

vascular disease, as well as to assess whether 

the high level of homocysteine is the determinant 

of its relationship with traditional and nontraditional 

risk factor in subjects with ASVD. 


Patients 

The study was designed as a cross-sectional 

study which included 99 patients with atherosclerotic 

vascular disease and 40 gender, age matched 

apparently healthy volunteers in the control 

group. Mean age of patients (64.0% males) was 

53.62±1.17 years and control subjects (56.0% 

males) was 57.49±1.71 years (p=0.08). ASVD in 

this study was defined as the presence of ischemic 

heart disease and history of angina, previous myocardial 

infarction, coronary artery bypass surgery 

or stenting, cerebrovascular event, transient ischemic 

attack, or peripheral vascular disease with 

or without amputation. Also, ASVD was confirmed 

by bilateral Carotid Intima-Media Thickness 

measurement. Pathological values were determined 

according to age and gender (17). For further 

analysis, ASVD group of patients was divided in 

two groups based on their tHcy level as: Normo- 

Hcy (normohomocysteinemic group of patients; 

n=28) and HyperHcy (hyperhomocysteinemic 

group of patients, n=71). The exclusion criteria 

127

128 


were diabetes mellitus, hypothyroidism, malignant 

disease, treatment with anticonvulsants, 

theophylline, or hormonal therapy which might 

influence Hcy levels. 

Written informed consent was obtained from 

all subjects. Investigations were carried out in 

accordance with the Declaration of Helsinki as 

revised in 2000. Ethical Committee of Medical 

School of the University of Sarajevo approved 

the experiment. 

Biochemical measurement 

Blood chemistry. Fasting blood samples were 

drawn from antecubital vein into siliconized 

tubes. Special care was taken with samples for 

homocysteine analysis, with tubes immediately 

placed on ice and transferred to the Clinical Chemistry 

and Biochemistry Centre of the University 

of Sarajevo. Blood samples for all analyses were 

centrifuged within 15 minutes at room temperature 

at 3000 rpm. Samples for tHcy analysis were 

kept at -80°C until the analyses. 

Lipid profile. Serum total cholesterol, highdensity 

lipoprotein cholesterol, HDLc and triglycerides 

were quantitatively measured by the 

Dimension Analyzer, DADE BEHRING using 

enzymatic methods. Reference intervals were 

3,1 - 5.2 mmol/L for total cholesterol, 1.04-1,55 

mmol/L for HDLc and 0,11- 1,7 mmol/L for triglycerides. 

LDLc was calculated by the Friedewald 

formula. Reference interval determinate by this 

method was 2,00 – 4,30 mmol/L. VLDLc cholesterol 

was calculated from triglycerides serum 

concentration and its reference interval was 0,13 

– 0,90 mmol/L. The atherogenic index (AI), a 

predictor of cardiovascular risk, was calculated 

using the following equation: LDLc / HDLc. Reference 

interval is 1,2 – 4,0. 

Uric acid. Uric acid was determined quantitatively 

by uricase method on Dimension, Dade 

Behring. Referral values for UA determined by 

this method were 208–428 μmol/L for males and 

155–357 μmol/L for females. 

hsCRP. High sensitivity CRP (hsCRP) was determined 

by means of particle enhanced immunonephelometry 

(BN Systems, Dade Behring, 

Marburg, Germany). The lower limit of the detection 

of this assay was 0.18mg/L. 

Homocysteine. Serum total homocysteine was 

measured by fluorescence polarization immuno- 

assay (Homocysteine; Abbott) on an automated 

analyzer (IMx system; Abbott). Optimal procedures 

in blood sample collection and handling 

were followed to prevent the passage of tHcy 

from red cells to plasma and thus ensure reliable 

measurements. The reference ranges for tHcy 

concentration established in our laboratory was 

5,9-16,0 μmol/L for males and 3,36-20,44 μmol/L 

for females. Hyperhomocysteinemia was defined 

as serum tHcy concentration ≥ 97,5 percentiles of 

our reference values which is 15,01 μmol/L for 

males and 12,44 μmol/L for females. 


Each value was expressed as the mean SEM or 

median and interquartile range where applicable. 

Differences in continuous variables between two 

groups were analyzed by the unpaired Student t 

test, while the difference between three groups 

of subjects (Control, NormoHcy and HyperHcy 

ASVD groups) was analysed with ANOVA or 

Kruskal-Wallis test where appropriate. P values 

less than 0.05 were considered statistically significant. 

Since tHcy, hsCRP and HDL were nonnormally 

distributed in this study, we normalized 

these data by log transformation before correlation 

analysis. Correlation coefficients were determined 

using Pearson correlation analysis. A stepwise 

multiple regression analysis was applied to 

examine the relationship between logtHcy and a 

set of clinical parameters including age, sex, lipids, 

hsCRP and UA. All statistical calculations 

were performed with the SPSS 16 software (version 

16.0, SPSS Inc, Chicago, Illinois, USA). 

RESULTS 

In ASVD group of subjects mean serum cholesterol, 

LDLc, VLDLc, HDLc concentration 

and atherogenic index was significantly lower 

compared to the control subjects (4.75±0.12 

vs. 6.13±0.12 mmol/L (p

Table 1. Mean serum lipids, uric acid and C-reactive protein 

concentration in control subjects, normohomocysteinemic 

and hyperhomocysteinemic subjects with atherosclerotic 

vascular disease* 

Control 

group 

(n=40) 

NormoHcy 

ASVD group 

(n=28) 

HyperHcy ASVD 

group (n=71) 

Cholesterol 

(mmol/L) 

6.13±0.12 5.62±0.19 4.4±0.13†‡ 

Triglycerides 

(mmol/L) 

2.17±0.18 1.85±0.12 2.00±0.17 

HDLc 

(mmol/L) 

1.12 

(0.96-1.36) 

0.93 

(0.79-1.09) 

0.89(0.73-1.08) 

LDLc 

(mmol/L) 

4.10±0.18 3.73±0.17 2.73±0.12†‡ 

VLDLc 

(mmol/L) 

1.18±0.19 0.95±0.09 0.79±0.04 

Atherogenic 

index 

4.4±0.25 4.9±0.4 3.2±0.18†‡ 

Uric acid 

(μmol/L) 

332.46±19.8 304±21.1 422.8±17.0†‡ 

CRP (mg/L) 1.8 (0.9-3.65) 

8.3 

(3.36-21.2) † 

4.2(1.08-4.2) 

*Data are presented as mean±SEM or median with interquartile 

range; ASVD, atherosclerotic vascular disease; normoHcy, normohomocysteinemic; 

HyperHcy, hyperhomocysteinemic; HDLc, high 

density lipoprotein cholesterol, VLDLc, very low density lipoprotein 

cholesterol; LDLc, low density lipoprotein cholesterol; 

†significant difference compared to control group (p

130 


lerotic vascular disease and its clinical manifestations 

(19). But apart from that, elevated level of 

homocysteine is considered as an independent risk 

factor for the development of atherosclerotic vascular 

disease. Elevated levels of tHcy have been 

found in patients with cardiovascular disease (20). 

Inside of the cells, homocysteine induces endoplasmic 

reticulum (ER) stress that activates genes 

encoding enzymes in the cholesterol and triglycerides 

biosynthesis and uptake pathways (21). 

In culture cells and murine models, Werstuck et 

al. have found significant increase in the content 

of cholesterol and triglycerides in the liver but 

not in plasma of hyperhomocysteinemic mice. A 

combination of increased endogenous cholesterol 

and triglyceride synthesis and LDL uptake, 

rather than decreased (impaired) hepatic export 

of VLDL lead to hepatic lipid accumulation in 

mice (22). Large number of case control and retrospective 

studies on patients have shown weak 

or strong positive correlation between tHcy and 

total cholesterol and LDLc (23). The results of 

our study have shown that hyperhomocysteinemic 

ASVD patients have a significantly lower 

concentration of cholesterol and LDLc compared 

to normohomocysteinemic ASVD patients and 

control subjects. Moreover, in hyperhomocysteinemic 

patients with ASVD there was a significant 

inverse association between logtHcy and cholesterol, 

VLDL, LDLc levels and AI. Association 

between logtHcy and cholesterol in normohomocysteinemic 

ASVD patients and in control group 

of subjects was not significant. Consistent with 

our findings, a study by Kalantar-Zadeh et al. has 

also demonstrated negative but not significant 

correlation between homocysteine and cholesterol 

(24). Hyperhomocysteinemia also enhances 

LDL uptake (19) which can explain a significant 

decrease in LDLc level, in our ASVD group of 

patients with hyperhomocysteinemia. 

HDL mediates reverse cholesterol transport and 

has protective, anti-inflammatory and antioxidative 

effects. A study by Holven et al. (25) indicated 

that hyperhomocysteinemic subjects have 

dysfunctional and reduced HDL levels which 

consequently impaired ability to induce cholesterol 

efflux from macrophages loaded with lipids. 

The mean values of HDLc was below reference 

interval and lower in the ASVD group of patients 

as compared to controls. In the study by Chan 

et al. (26) HDLc concentration in subjects with 

cardiovascular disease was significantly lower, 

Figure 2. Relationship between log total homocysteine (tHcy) and serum cholesterol (A), VLDLc (B), LDLc (C) and atherogenic 

index (D) in hyperhomocysteinemic patients with atherosclerotic vascular disease

while no significant differences in LDL and triglycerides 

concentration compared to controls was 

observed. They suggested that hyperhomocysteinemia 

is not influenced by lipid profile. 

Malanovic et al. suggested that VLDLc synthesis 

is decreased in hyperhomocysteinemic state 

(27). Accumulation of S-adenosyl-homocysteine 

decreases synthesis of phosphatidylcholine, main 

phospholipids for VLDL synthesis. Our results 

are in concordance with the aforementioned study 

confirming that VLDLc was significantly lower 

in ASVD subjects as compared to controls, while 

negative correlation of logtHcy and VLDLc was 

found in hyperhomocysteinemic subjects with 

atherosclerosis. This finding might be explained 

by the homocysteine mediating decrease of the 

VLDL output from the liver. 

Homocysteine increases cholesterol and triglycerides 

production and its accumulation in endothelial 

cells may be a link between elevated plasma 

homocysteine and development of atherosclerosis 

(28). Accumulation of lipid in tissues that are 

sensitive to ER stress can be an explanation as to 

Figure 3. Relationship between log total homocysteine (tHcy) 

and serum uric acid (A) and logCRP in normohomocysteinemic 

patients with atherosclerotic vascular diseases 


why patients with hyperhomocysteinemia have a 

relatively normal serum lipid profiles and no positive 

correlation between elevated plasma tHcy 

and triglycerides. 

Many studies have reported a direct relationship 

between Hcy as cardiovascular risk factor and lipids 

in patients with cardiovascular disease (23,29). 

Our results, on the contrary, have shown an inverse 

relation between elevated Hcy level and lipids as 

traditional risk factors but the mechanism responsible 

for this can not be explained by this study. 

In our study we have observed significantly higher 

serum uric acid in patients with ASVD as compared 

to control subjects, but this finding could only 

be attributed to the ASVD patients with hyperhomocysteinemia, 

while in normohomocysteinemic 

patients serum UA level was similar to the level 

observed in the control subjects. The results of our 

study have shown that serum homocysteine level 

within the reference range was strongly independently 

associated with serum uric acid level in 

patients with ASVD, while in patients with hyperhomocysteinemia 

such a relationship could not be 

confirmed. Our results are in concordance with 

the studies which also confirmed a direct relation 

between plasma homocysteine levels and serum 

UA in patients with atherosclerosis and in control 

subjects (30, 31). Possible mechanisms underlying 

these associations are both the preference of adenosine 

which has originated from S-adenosyl-homocysteine 

for the incorporation into a precursor 

pool for uric acid and consequent syntheses of 

homocysteine and UA and possible compensatory 

response counteracted excessive oxidative stress 

in subjects with ASVD with higher UA levels 

(32). However, the role of UA in humans is still 

uncertain. Uric acid in the early stages of atherosclerotic 

process is known to act as an antioxidant 

and may be one of the strongest determinants of 

plasma antioxidative capacity (33). However, later 

in the atherosclerotic process when UA levels are 

elevated this previous antioxidant paradoxically 

becomes prooxidant (33). 

A direct relation between homocysteine and UA 

levels is known to occur in patients with atherosclerosis 

(34). Not only do these two track together (possibly 

reflecting an underlying elevated tension of 

redox stress) but also may be synergistic in creating 

an elevated tension of redox stress, especially in the 

rupture prone, vulnerable atherosclerotic plaque 

with depletion of local occurring antioxidants (35). 

131

132 


The limitations of the study could be the relatively 

small number of ASVD patients with normohomocysteinemia 

included in the study which 

might have influenced the results on association 

between tHcy and risk factors. The ASVD is a 

wide term including different kinds of diseases 

which also might have the influence on the observed 

association between tHcy and traditional and 

nontraditional risk factors. Nontraditional risk 

factors should be combined with conventional 

risk factors for detecting and monitoring people 

in risk for atherosclerotic vascular disease. Ba- 

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This study was partially funded by a grant from 

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Povezanost homocisteina s tradicionalnim i netradicionalnim faktorima rizika kod 

pacijenata s aterosklerotskom vaskularnom bolesti 

Emina Kiseljaković1 , Amina Valjevac2 , Sabaheta Hasić1 , Emina Nakaš-Ićindić2 , Alen Džubur3 , Radivoj 

Jadrić1 1 2 3 Katedra za medicinsku biohemiju, Katedra za fiziologiju čovjeka; Medicinski fakultet, Univerzitet u Sarajevu; Klinika za bolesti srca i 

reumatizma, Klinički centar Univerziteta u Sarajevu; Bosna i Hercegovina 

SAŽETAK 

Cilj Ispitati povezanost homocisteina s tradicionalnim i netradicionalnim faktorima rizika kod pacijenata 

s aterosklerotskom vaskularnom bolesti (ASVB). 

Metode U ovu presječnu studiju bilo je uključeno 90 pacijenata s ASVB-om i 40 prividno zdravih, kontrolnih 

pacijenata, kojima smo određivali lipidni profil, visoko osjetljivi C reaktivni protein (hsCRP), 

mokraćnu kiselinu i ukupni homocistein (tHcy). 

Rezultati Srednja vrijednost koncentracije tHcy bila je značajno veća u ASVB grupi pacijenata u odnosu 

na kontrolu (18.7 (13.65-24.45) vs. 11.48 (10.03-14.2) µmol/L (p

134 


Coagulation factor VIII activity in diabetic patients 

Nermina Babić 1 , Amela Dervišević 1 , Jasminko Huskić 1 , Miralem Musić 2 

1 Department of Physiology; 2 Department of Pathophysiology; Medical Faculty, University of Sarajevo, Sarajevo, Bosnia and Herzegovina 


Nermina Babić 

Department of Physiology, 





Phone: +387 33 226 472; 

fax.: +387 33 203 670 

E-mail: nerminab@yahoo.com 


2010; 


2010; 

Accepted: 


ABSTRACT 


Aim To examine coagulation factor VIII activity in plasma, as a 

risk factor for thrombosis, in the patients with diabetes mellitus 

(DM). Also, to assess its relationship with fibrinogen and fasting 

blood glucose concentrations and with body mass index. 

Methods The plasma coagulation factor VIII activity, plasma levels 

of fibrinogen and blood glucose concentrations were measured 

in 30 patients with DM type 1, 30 patients with DM type 2 and 

in 30 healthy subjects. Body weight and body height were also 

measured and BMI was calculated. 

Results The plasma factor VIII activity in patients with DM type 

1 and patients with DM type 2 was significantly higher than the 

values measured in healthy subjects. There was no significant difference 

in the factor VIII activity between patients with DM type 1 

and type 2. The concentrations of fibrinogen and blood glucose in 

both groups of patients were significantly higher than in the group 

of healthy subjects. Patients with DM type 2 had a significantly 

higher BMI compared to healthy subjects, as well as compared to 

patients with DM type 1. There was a significant positive correlation 

between plasma factor VIII activity and plasma level of fibrinogen 

and a significant negative correlation between factor VIII 

activity and BMI in patients with DM type 2. 

Conclusion Diabetic patients have the elevated plasma coagulation 

factor VIII activity and increased fibrinogen concentration thus 

an increased risk of thrombosis and vascular diseases. 

Key words: coagulation factor VIII, thrombosis, diabetes melitus

INTRODUCTION 

Diabetes mellitus is associated with an increased 

risk of atherosclerosis, and macrovascular complications 

are a major cause of morbidity and mortality 

in this disease (1). Thrombosis is the cause of 

death in 80% of patients with diabetes (2). 

Endothelial dysfunction is the earliest event 

that precedes the development and progression 

of diabetic vascular complications (3). The pathogenesis 

of endothelial dysfunction in diabetes 

is complex. Multiple cellular and molecular 

mechanisms are involved in the development of 

diabetic dysfunctional endothelium (e.g. hyperglycemia, 

insulin resistance, impaired lipid metabolism 

and lipoproteins, oxidative stress) (1, 4). 

The plasma levels of some biomarkers may be 

measured as indirect indices of endothelial cell 

damage, activation and inflammation to assess 

endothelial function (nitric oxide, asymmetric 

dimethylarginine, endothelin-1, von Willebrand 

factor, adhesion molecule, plasminogen activator 

inhibitor-1) (5-7). 

The plasma levels of many clotting factors, including 

fibrinogen, factor VII, factor VIII, factor 

XI, factor XII, kallikrein and von Willebrand 

factor, are elevated in diabetes. The fibrinolytic 

system is relatively inhibited as a consequence 

of an increase in plasminogen activator inhibitor 

type-1 levels (2). This procoagulant state and hypofibrinolysis 

contributes to macrovascular and 

microvascular complications of diabetes mellitus 

(8,9). 

In 1980 a prospective study indicated factor VIII 

to be a risk factor for arterial disease (10) and other 

studies also suggested association of elevated 

factor VIII with both cardiac and cerebral vascular 

disease (11,12). Several studies showed that 

elevated plasma levels of factor VIII are associated 

with an increased risk of venous thrombosis 

(13,14,) and the risk of recurrent venous thromboembolism 

(15). 

Based on these observations, we have assumed 

that diabetic patients may have an increased risk 

of thrombosis, if their coagulation factor VIII activity 

is elevated. 

This study was designed to examine coagulation 

factor VIII activity in plasma, as a risk factor 

for thrombosis, in patients with diabetes mellitus 

(DM). Its purpose was also to assess its relati- 

Babić et al Factor VIII activity in diabetic patients 

onship with fibrinogen and fasting blood glucose 

concentrations and with body mass index (BMI). 


The study population consisted of 30 patients with 

type 1 diabetes and 30 patients with type 2 diabetes. 

The control group consisted of 30 age- and 

sex- matched healthy subjects. Subjects with manifest 

cardiovascular disease, peripheral vascular 

disease or cerebrovascular accident, history of coagulation 

disorder, liver dysfunction, malignancy, 

receiving medications that could affect the coagulation-fibrinolytic 

system such as antiplatelet 

agents, anticoagulants, oral contraceptives etc. 

were excluded from the study. The Ethical Commitee 

of our institution approved the protocol of 

this study. All participants were informed about 

the study and signed informed consent forms. 

Blood samples were collected after an overnight 

fasting. Glucose levels were determined by the 

glucose oxidase method in serum. For coagulation, 

a venous blood sample (9 ml) was collected 

into special vacutainer tubes containing 3,2% sodium 

citrate. Factor VIII activity was determined 

by standard coagulometric method by using coagulation 

factor VIII deficient plasma (reagent) on 

automatic analyzer (Behring Coagulation Timer, 

BCS, Dade Behring). Plasma fibrinogen was measured 

by clot method using the BCS Coagulation 

System (Dade Behring). 

The normal ranges for measured parameters were 

factor VIII activity 70-150%, plasma fibrinogen 

5,3-10,3 mmol/L, fasting blood glucose 3,3-6,1 

mmol/L. 

Body weight was measured using a portable stadiometer, 

and weight was assessed for each participant 

in light clothing without shoes using the 

electronic scale. Body mass index (BMI = weight 

(kg)/height (m2 )) was calculated based on measured 

weight and height. 

The test results were statistically processed using 

descriptive statistics and for each study group 

mean ( X ), standard deviation (SD) and standard 

error of the mean (SEM) were determined. 

To determine statistically significant differences 

between the groups Student t-test was used. A level 

of correlation was determined by Spearman 

method. Probability values less than 0,05 were 

considered significant. 

135

136 


RESULTS 

The plasma factor VIII activity of patients with 

DM type 1 (141,48±5,59 % of norm.; 8±SEM) 

and patients with DM type 2 (134,36±5,15) was 

significantly higher than the values measured in 

healthy subjects (116,74±4,03; p

uting factor for the occurrence of vascular complications 

in diabetes (18). 

Elevated coagulation factor VIII activity is independent 

risk factor for thrombosis, with a greater 

impact on venous than arterial thrombosis (13). 

The results of this study have shown that the plasma 

factor VIII activities were significantly higher 

in both groups of the patients than in the controls, 

but within the normal range of our laboratory. There 

was no statistically significant difference in the 

plasma factor VIII activity between patients with 

DM type 1 and DM type 2. Various researchers 

have reported elevated factor VIII activity in diabetic 

patients, mostly in patients with DM type 2 

(9, 19). Erem et al. (18), Madan et al. (8) found no 

differences in plasma factor VIII activity between 

patients with DM type 2 and control. Both groups 

of researchers have studied coagulation and fibrinolysis 

parameters in type 2 diabetic patients with 

and without vascular complications. 

The mechanism by which factor VIII influence 

thrombotic risk has not yet been identified. The 

most factor VIII circulates as a complex with von 

Willebrand factor (vWf) (7,20) High shear stress, 

such as those that occur in stenosed vessels, 

increase vWf secretion by vascular endothelium 

and, thus, will stimulate platelet adhesion and aggregation 

on the damaged endothelium (7). The 

main function of factor VIII is to activate factor 

X functioning as a cofactor for activated factor 

IX in the presence of phospholipids and calcium 

(20). Elevated factor VIII induces the increase 

of thrombin formation and may contribute to the 

developement of large occlusive thromb. Factor 

VIII activity level above 100-150 IU/dL(100%- 

150%) increases 3-fold the risk of thrombosis 

while the level above 150 IU/dL (150%) increases 

the same risk 6-fold when compared with 

levels below 100% (13). Furthermore, each increase 

in factor VIII level with 10 IU/dL (10%) 

is associated with a 10% increase in the risk of 

the thrombotic event (12). 

The role of fibrinogen in prediction of thrombosis 

has not been established yet. Some studies report 

that patients with high plasma fibrinogen level 

have an increased risk of venous thrombosis (21). 

On the other hand, in the LITE study elevated fibrinogen 

levels did not predict an increased risk 

of venous thromboembolism (22) The results of 

this study have shown that factor VIII and von 


Willebrand factor were linearly associated with 

an increased risk of venous thromboembolism 

whereas an elevated factor VII level was a possible 

risk factor. 

In our study, the plasma fibrinogen concentrations 

were found to be significantly higher in diabetic 

patients than in the control subjects. Our findings 

are in accordance with the results of Madan et al 

(8) and Erem et al (18). However, these authors 

measured coagulation parameters only in type 2 

diabetic patients. 

Fibrinogen is required for platelet aggregation 

and hyperfibrinogenaemia was related to the 

risk of venous thrombosis (21,23). A significant 

positive correlation between plasma factor VIII 

activity, as a key procoagulant enzymatic cofactor 

and plasma concentration of fibrinogen in patients 

with DM type 2 was found in our study. 

This relationship may contribute to a higher risk 

of thrombosis in diabetic patients. 

The results of this study have shown that BMI of 

type 2 diabetic patients was significantly higher 

than BMI of type 1 diabetic patients and control 

subjects. Patients with DM type 2 and abdominal 

fat pattering and, also obese individuals displayed 

higher plasma activites of factor VIII (24). Adipose 

tissue dysfunction could thus play a causal 

role in the prothrombotic state observed in obesity, 

by directly or indirectly affecting coagulation 

and fibrinolysis. Hepatic synthesis of the coagulation 

factors fibrinogen, factor VII, factor VIII 

and tissue factor are stimulated by pro-inflammatory 

cytokines originating in the visceral adipose 

tissue (19, 24). According to the literature it was 

expected that the increased activity of factor VIII 

positively correlated with BMI. However, in our 

study plasma factor VIII activity was negatively 

correlated with BMI in type 2 diabetic patients. 

We could not explain these results. Perhaps the 

study should include a larger number of subjects. 

Factor VIII is an acute phase reactant and its levels 

can be affected by many factors, including 

vWf level and blood type. This study has not examined 

vWf activity in diabetic patients and blood 

types of the patients. 

Generally, the areas of visceral adipose tissue 

assessed by using computed tomography seems 

to be more associated with plasma hemostatic parameters 

than BMI. 

137

138 


In our study the patients with DM had elevated 

plasma coagulation factor VIII activity, but within 

normal range of our laboratory and increased 

fibrinogen concentration. Also, the results have 

shown that type 2 diabetic patients have increased 

BMI. According to the results obtained and 

data in the literature it has been concluded that 

this procoagulant state may contribute to the increased 

risk of thrombosis and vascular disease 

in diabetic patients. 

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1. Pandolfi A, De Filips EA. Chronic hyperglicemia 

and nitric oxide bioavailability play a pivotal role in 

pro-atherogenic vascular modifications. Genes Nutr 

2007; 2:195-208. 

2. Carr ME. Diabetes mellitus: A hypercoagulable state. 

J Diabetes Complications 2001;15:44-54. 

3. De Vriese AS, Verbeuren TJ, Van De Voorde J, Lameire 

NH, Vanhoutte PM. Endothelial dysfunction 

in diabetes. Br J Pharmacol 2000;130:963-74. 

4. Guerci B, Bőhme P, Kearney-Schwartz A, Zannad 

F, Drouin P. Endothelial dysfunction and type 2 diabetes. 

Diabetes Metab 2001; 27:436-47. 

5. Hamilton SJ, Chew GT, Watts GF. Therapeutic regulation 

of endothelial dysfunction in type 2 diabetes 

mellitus. Diabetes Vasc Dis Res 2007; 4:89–102 

6. Thorand B, Baumert J, Chambless L, Meisinger C, 

Kolb H, Döring A, Löwel H, Koenig W, the MO- 

NICA/KORA Study Group. Elevated markers of 

endothelial dysfunction predict type 2 diabetes mellitus 

in middle-aged men and women from the general 

population. Arterioscler Thromb Vasc Biol 2006; 

26:398-405. 

7. Babic N, Huskic J, Nakas-Icindic E, Tihic-Kapidzic 

S, Music M. New information about von 

Willebrand`s factor? Health Med 2010; 4:187-94. 

8. Madan R, Gupta B, Saluja S, Kansra UC, Tripathi 

BK, Guliani BP. Coagulation profile in diabetes and 

its association with diabetic microvascular complications. 

JAPI 2010; 58:481-4. 

9. Dunn EJ, 

Grant PJ. Type 2 diabetes: an atherothrombotic 

syndrome. Curr Mol Med 2005; 5:323-32. 

10. Koster T, Blann AD, Briet E, Vandenbroucke JP, 

Rosendaal FR. Role of clotting factor VIII in effect 

of von Willebrand factor on occurrence of deep-vein 

thrombosis. Lancet 1995; 345:152-5. 

11. O’Donnell J, Laffan M Elevated plasma factor VIII 

levels-a novel risk factor for venous thromboembolism. 

Clin Lab 2001; 47(1-2):1-6. 

12. Kraaijenhagen RA, in’t Anker PS, Koopman MMW, 

Reitsma PH, Prins MH, Van Den Ende A, Büller HR. 

High plasma concentration of factor VIIIc is a major 

risk factor for venous thromboembolism. Thromb 

Haemost 2000; 83:5-9. 

13. Kamphuisen PW, Eikenboom CJ, Bertina RM. Elevated 

factor VIII levels and the risk of thrombosis. 

Arterioscler Thromb Vasc Biol 2001;21:731-73. 



by Nermina Babić, Jasminko Huskić, Suzana 

Tihić-Kapidžić, Amela Dervišević. Factor 

VIII as a marker of higher risk for thrombosis development 

in diabetes mellitus patients. Proceedings 

of the 1st Congress of Medical Biochemists 

of Bosnia and Herzegovina with international 

participation, Sarajevo/Bosnia and Herzegovina, 

May 21-22 2010. Programme & Abstracts, Association 

of medical biochemists in Bosnia and 

Herzegovina, Sarajevo, 2010, P51. 


14. Mettinger KL. A study of hemostasis in ischemic cerebrovascular 

disease, I: Abnormalities in factor VIII 

and antithrombin. Thromb Res 1982; 26:183-92. 

15. Kyrle PA, Minar E, Hirschl M, Bialonczyk C, Stain 

M, Schneider B,Weltermann A, Speiser W, Lechner 

K, Eichinger S. High plasma levels of factor VIII 

and the risk of recurrent venous thromboembolism. 

N Engl J Med 2000; 343:457–62. 

16. Grant P. J. Diabetes mellitus as a prothrombotic condition. 

Journal of Internal Medicine 2007,262:157– 

72 

17. Bugnicourt JM, Roussel B, Tramier B, Lamy Chl, 

Godefroy O. Cerebral venous thrombosis and plasma 

concentrations of factor VIII and von Willebrand 

factor: a case control study J Neurol Neurosurg 

Psychiatry 2007; 78:699–701. 

18. Erem C, Hacıhasanog˘lu A, Çelik Ş, Ovalı E, Ersöz 

HÖ, Ukinç K, Deger O, Telatar M. Coagulation and 

fibrinolysis parameters in Type 2 diabetic patients 

with and without diabetic vascular complications. 

Med Princ Pract 2005;14:22–30. 

19. Coca M, Cucuianu M, Hancu N. Effect of abdominal 

obesity on prothrombitic tendency in type 2 diabetes. 

Behavior of clotting factors VII and VIII, fibrinogen, 

and von Willebrand factor. Rom J Intern Med 

2005; 43:115-26. 

20. Hultin MB. Role of human factor VIII in factor X 

activation. J Clin Invest 1982; 69(4): 950–958. 

21. Koster T, Rosendaal FR, Reitsma PH, van derVelden 

PA, Briët E, Vandenbroucke JP. Factor VII and 

fibrinogen levels as risk factors for venous thrombosis. 

A case-control study of plasma levels and DNA 

polymorphisms - the Leiden Thrombophilia Study 

(LETS). Thromb Haemost 1994;71:719-22. 

22. Tsai AW, Cushman M, Rosamund WD, Heckbert 

SR, Tracy RP, Aleksic N, Folsom AR. Coagulation 

factors, inflammation markers, and venous thromboembolism: 

the longitudinal investigation of thromboembolism 

etiology (LITE). Am J Med 2002; 

113:636-42. 

23. de Moerloose P, 

Boehlen F, Neerman-Arbez M. Fibrinogen 

and the risk of thrombosis. Semin Thromb 

Hemost 2010;36(1):7-17. 

24. Faber DR, 

de Groot PG, Visseren FL Role of adipose 

tissue in haemostasis, coagulation and fibrinolysis. 

Obes Rev 2009; 10:554-63.

Aktivnost VIII faktora koagulacije u pacijenata s diabetes mellitusom 

Nermina Babić 1 , Amela Dervišević 1 , Jasminko Huskić 1 , Miralem Musić 2 

1 Katedra za fiziologiju; 2 Katedra za patofiziologiju; Medicinski fakultet, Univerzitet u Sarajevu, Sarajevo, Bosna i Hercegovina 

SAŽETAK 

Cilj Ispitati aktivnost VIII faktora koagulacije u plazmi, kao faktora rizika za razvoj tromboze, kod 

pacijenata s diabetes mellitusom (DM). Također, ispitati povezanost aktivnosti VIII faktora koagulacije 

s koncentracijama fibrinogena i glukoze u krvi i indeksom tjelesne mase (BMI). 

Metode U studiju su bili uključeni pacijenti s DM tip 1 (N=30) i tip 2 (N=30), kao i zdravi ispitanici 

(N=30). Svim ispitanicima izmjerena je aktivnost VIII faktora koagulacije u plazmi, koncentracije fibrinogena, 

glukoze, tjelesna masa i tjelesna visina, te određen BMI. 

Rezultati Aktivnost VIII faktora u plazmi pacijenata s DM tip 1 i tip 2 bila je statistički signifikantno 

veća u odnosu na vrijednosti izmjerene kod zdravih ispitanika. Nije bilo signifikantne razlike u aktivnosti 

VIII faktora između pacijenata s DM tip 1 i tip 2. Koncentracija fibrinogena i glukoze u krvi, kod obje 

grupe pacijenata, bila je statistički signifikantno veća u odnosu na grupu zdravih ispitanika. Pacijenti s 

DM tip 2 imali su signifikantno veći BMI u odnosu na zdrave ispitanike, kao i u odnosu na pacijente s 

DM tip 1. Utvrđena je značajna pozitivna korelacija između aktivnosti VIII faktora i fibrinogena, kao i 

značajna negativna korelacija između aktivnosti VIII faktora i BMI u pacijenata s DM tip 2. 

Zaključak Pacijenti s DM imaju povećanu aktivnost VIII faktora koagulacije i koncentraciju fibrinogena, 

te time i povećan rizik od razvoja tromboze i vaskularnih oboljenja. 

Ključne riječi: VIII faktor koagulacije, tromboza, diabetes melitus 

Original submission: 2010; Revised submission: 2010; Accepted: 25 November 2010. 


139

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Time-dependent responses of rat troponin I and cardiac injury 

following isoproterenol administration 

Sabaheta Hasić 1 , Radivoj Jadrić 1 , Emina Kiseljaković 1 , Amina Valjevac 1 , Zakira Mornjaković 2 , Mira 

Winterhalter-Jadrić 1 

1 Institute for Physiology and Biochemistry, 2 Institute for Histology and Embryology; Medical Faculty, University of Sarajevo, Sarajevo, 



Sabaheta Hasić, 

Institute for Physiology and Biochemistry, 

University Sarajevo, Medical Faculty, 



Phone: +387 33 663 743; 

fax: +387 33 203 670 

E-mail: sabaheta.hasic@mf.unsa.ba 





Accepted: 


ABSTRACT 


Aim To develop a rat model of myocardial infarction induced by 

isoproterenol (ISO). We investigated a type of histological myocardial 

changes and cardiac troponin I (TnI) kinetic. 

Methods The study has used adult, male, Wistar strain rats. Rats 

were distributed in ISO and control groups. Rats treated with ISO 

were divided into groups according to the time of cTnI and myocardial 

lesion analyses: ISO I (30’), ISO II (60’), ISO III (120’) 

and ISO IV (240’). We determined cTnI (Life Diagnostics Inc. 

West Chester PA, USA) in the serum by ELISA method. We performed 

histological analysis on the specimens of left ventricular 

wall stained by hematoxillin-eosin (HE) method. 

Results The first statistically significant rise of cTnI was noted 30 

minutes after the ISO administration. There was no statistically 

significant difference between cTnI mean values among the ISO 

groups. Observed myocardial histological changes were time dependent. 

Conclusions This model can be suitable for cardioprotective and 

cardiotoxicity supstance investigations followed by cTnI measurement 

in blood. The similarity between induced myocardial lesion 

on animal model in our study and human myocardial lesion in ischemia 

give us sufficient impulse for further preclinical researches 

of new cardiac markers. 

Key words: cardiac troponin I, isoproterenol, heart, rat

INTRODUCTION 

Cardiovascular diseases are one of leading causes 

of mortality and morbidity (1). Animal model 

of myocardial infarction is very important for the 

development of new approaches in prevention, diagnosis 

and therapy of human myocardial infarction. 

Animal models are also used in new drugs 

investigation and testing of their cardioprotective/ 

cardiotoxicity effects (2). More easily applicable 

noninvasive methods of myocardial damage 

induction are used in small laboratory animals. 

At the beginning of the twentieth century, it was 

known that adrenaline might be a cause of coronary 

heart disease of non-atherogenic origin. The 

catecholamines, as the beta-receptor agonists, cause 

focal myocardial necroses through their hemodynamic 

effects. ISO is the first synthetic catecholamine 

used in the therapy of bronchial asthma 

since 1940. (3). Extrapulmonary side effects such 

as tachycardia, arrhythmias, and palpitations are 

noted as consequences of ISO non selectivity. The 

main mechanisms of myocardial damage induced 

by ISO include alteration of coronary microcirculation, 

cardiomyocyte membrane permeability 

changes, excessive accumulation of calcium ions 

in cardiomyocyte, myocardial oxidative stress, 

lipolysis induction and auto-oxidation of catecholamine 

in aminochrome (4-7). Cardiac TnI, contractile 

protein of heart muscle has been a gold 

standard since 2000 for diagnosing myocardial 

damage (8). Cardiac troponins are predominantly 

bound to actin filaments of sarcomeres, with a 

small proportion of cTnI 3–8% found in the soluble 

cytoplasmic pool (9). 

This study has investigated whether a dose of 100 

mg/kg of rat body weight (b.w.) was sufficient to 

induce rat myocardial damage and evaluated the 

importance of cTnI as a biomarker of myocardial 

damage on animal model. Moreover, it has evaluated 

a type of histological myocardial changes 

induced by the ISO administration. It has hypothesized 

that the ISO administration is associated 

with increased concentration of circulating cTnI 

and that increased serum concentrations of cTnI 

are associated with rat myocardial damage. 

MATERIAL AND METHODS 

Thirty adult male Wistar rats of average b.w. 277 

g, ± 4.03 g were used for the study. They were 

raised and housed in air-conditioned, humidity- 

Hasić et al TnI and cardiac injury 

controlled cages. Rats had free access to water 

and commercial food during the experimental period. 

Ethical Committee of the School of Medicine, 

University of Sarajevo, approved the experiment. 

ISO dose 100mg/kg of rat b.w was used 

for administration to the ISO groups and 0,95% 

NaCl for the control group. Isoproterenol hydrochloride 

was manufactured by Sigma Chemical 

Company, USA. 

The rats were divided in two groups: isoproterenol 

group (ISO; n=24) and control group (CG; 

n=6). Blood was drawn from the conscious animals 

from the tail vein before the ISO or saline 

injection to determine basal cTnI blood level 

(controls). The ISO rats were treated with single 

subcutaneous (s.c.) dose of ISO (100 mg/kg b.w. 

dissolved in saline). The control group rats were 

treated with 0, 95% NaCl by the same route. We 

have administered subcutaneously a volume of 

solution (ISO dissolved in saline for ISO groups 

or saline for control group) that corresponds to 

the species and animals’ body weight (10). The 

ISO treated rats were divided into groups according 

to the time of cTnI and myocardial lesions 

analysis: ISO I (30 minutes), ISO II (60 minutes), 

ISO III (120 minutes) and IV group (240 minutes), 

each group consisting of 6 rats. After the 

injection of ISO or saline solution, blood samples 

were taken from abdominal aorta after the 

scheduled time for each group. Blood and heart 

samples were taken from ether-anaesthetized rats 

and then the rats were euthanized by decapitation. 

The blood was centrifuged for 10 minutes 

at 4000 r.p.m. The sera were frozen and stored 

at -20° until determination. cTnI was determined 

using an enzyme-linked immunosorbent assay 

(ELISA) developed by Life Diagnostics Inc. West 

Chester PA, USA, HIGH SENSITIVITY RAT 

CARDIAC TROPONIN-I ELISA KIT. We used 

immunoanalyzer STAT FAX 2100, USA. Values 

of cTnI are given in ng/ml. The lower limit 

of detection was 0,156 ng/mL. Rat hearts were 

dissected for histological examination, shortly 

after blood samples were taken. Left ventricular 

tissue was placed in 10% buffered formalin solution, 

embedded in paraffin, sectioned at 4-5 µm 

and stained with hematoxylin-eosin and observed 

microscopically. We performed a qualitative histological 

analysis using microscope Nikon type 

400E with a digital camera. 

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142 


We presented data as mean ± SEM. Nonparametric 

Kruskall-Wallis was used for testing 

between-group differences. A post hoc comparison 

was performed by the Mann-Whitney U test. 

Comparison of the two means of cTnI before and 

after ISO or saline application was carried out by 

using paired t-test. We considered p value less 

than 0.05 (p0.05). 

The mean values of cTnI measured in all rats 

were given in Figure 1. This figure presents cTnI 

value in groups before and after ISO/saline ad- 

ministration. Mean value of ISO III cTnI was 6.36±0.64 ng/ml. 

At the four–hour interval cTnI was 7.51± 0.18. 

We noted the highest cTnI serum value in group 

ISO IV and the lowest one in ISO III group. 

Figure 3 (A) illustrates the section of normal 

myocardial tissue in control rats. In ISO rats, the 

change of the subendocardial layer and perivascular 

cardiomyocytes was more intense as compa- 

Figure 1. The mean cTnI value in experimental groups* 

*values are given as mean±SEM. † p

ed with the change of subepicardial layer. Histological 

examinations of the myocardial tissues in 

ISO I and II rats (Figure 3 B and C) revealed acute 

extensive myofibrillar degeneration. The range 

of changes varied from myofibrillar eosinophilia 

with the loss of the normal striation pattern, presentation 

of hypercontraction bands, oedema, vacuolisation, 

granular degeneration to hyperchromatic 

nuclei with inhomogeneous content. 

Myocardial histological changes of ISO III and IV 

(D and E) were in form of coagulative necrosis. 

There were cardiomyocytes fragmentation and 

nuclei disappearance. Nuclear vacuolisation and 

myofibrillar lysis were complete in the more severe 

forms of lesion. Nuclei of these areas were in 

phases of pyknosis, lysis or complete disappearance. 

The usual cellular arrangement disappeared. 

DISCUSSION 

The analysis of cardiac troponin I is considered 

to be the “gold standard“ for the non-invasive diagnosis 

of myocardial injury in people and small 

animals. Concentration of cardiac injury biomarkers 

can be altered in samples collected from 

deeply sedated animals as a result of generalized 

hypoxia (11). In our study, we used light ether 

anesthesia shortly before sampling to avoid anesthesia 

induced generalized hypoxia. Cardiac TnI 

has replaced traditionally used cardiac biomarkers 

such as creatine-kinase and its isoenzymes 

due to its high sensitivity and specificity for the 

detection of myocardial injury (12). In our study, 

cardiac TnI blood level in control rats was bellow 

the detection limit of used immunoassay. Subcutaneous 

saline administration to rats induced undetectable 

cTnI changes in blood. Sensitivity of 

ELISA Life Diagnostic immunoassay used in our 

study was 0.156 ng/ml. In investigation of Kurata 

and associates performed on rat myocardial damage 

induced by ISO did not detect cTnI before 

and after saline application (13). They used the 

same immunoassay (Life Diagnostics). A rise of 

blood cTnT in the control group of rats, as in the 

study of Bertichant and associates, demonstrates 

possibility of minimal myocardial damage induced 

by stress tachycardia after saline application. 

In the same study, there were no significant serum 

cTnI changes in comparison with its basal 

values (14). Except during myocardial necrosis 

development, intracellular cTnI degradation and 


subsequent release of its degradation products in 

blood occur during ischemia. Mechanical stretch 

of cardiomyocytes during pressure or volume 

overload, initiates a cascade of intracellular signals, 

including increased intracellular calcium 

concentration and nitric oxide formation, and the 

activation of intracellular proteases (15,16,17). 

We noted a biphasic kinetic of cTnI because of 

the reduction of serum cTnI in the ISO III group. 

Biphasic kinetic indicates the release of free 

cytosol cTnI and its rapid excretion from the 

circulation. There was no significant difference 

between serum values of cTnI among the ISO 

groups. It indicates the slight proteolytic degradation 

of myofibrillar apparatus and a continuous 

cTnI release into the circulation. By degradation 

of contractile apparatus, a complexed form of 

cTnI is mainly released into the circulation (TnI- 

TnT-TnC). The half-life of complexed form is 

about 2 hours (18). An impaired renal clearance 

of cTnI caused by the impaired renal function 

explains a new cTnI increase in blood of ISO 

IV rats. An impaired glomerular filtration rate in 

AMI patients is caused by impaired myocardial 

function (19,20). A continuous releasing of cTnI 

from myocardial lesions and impaired renal function 

are possible reasons for a lack of significant 

difference in cTnI between the ISO groups. In the 

study of O’Brien and associates, cTnI reached 

peak level at 4-6 hours after the ISO application. 

Our findings are in accordance with their results 

(21). Cardiac TnI increase in blood and severity 

of myocardial histological changes depend on 

dose and route of ISO application (14,22,23). 

We noted a normal myocardial histological structure 

in the control group of rats. In other studies, 

rats from the control group exhibited histological 

sign of mild myocardial infarction. Histological 

myocardial changes were caused by hypoperfusion 

and tachycardia induced by stress (23,24). Our 

results indicate time dependency of histological 

changes induced by ISO. Changes were more intensive 

in subendocardium and around the blood 

vessels compared with other myocardial portions. 

The distribution of histological changes is a consequence 

of ISO influence on adrenergic receptors 

followed by blood pressure reduction and myocardial 

necrosis development through hypoxia of 

myocardial tissue (22). After two hours a clear 

temporal disconnect occurred between myocar- 

143

144 


dial lesion developing and declining serum cTnI 

concentration. We have noted that release of cTnI 

begins early in myocardial injury, prior to necrosis 

development as in the studies of York and 

associates and Clements and associates (22, 25). 

Serum troponin values reflect the development of 

histopathologic lesions (22,25). 

The adrenergic stimulation causes an enhanced 

transmembrane calcium influx, which results in 

an increase in the rate and force of contraction. 

That leads to an increase in the energy and oxygen 

requirements. A decrease in the duration of 

diastole induced by tachycardia results in hypoxia 

development in the least perfused area of 

the myocardium-subendocardial area. Since the 

coronary pressure decreases, the perfusion pressure 

is the lowest in this zone (26). 

A general limitation of this study is a lack of immunoassays 

standardisation for cTnI, as in human 

studies, and impossibility of comparison of 

our results with results of other studies that used 

the test from different manufacturers. 

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This model can be suitable for cardioprotective 

and cardiotoxicity substance investigations followed 

by cTnI measurement in blood. The similarity 

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animal model in our study and human myocardial 

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for further preclinical researches of new cardiac 

markers. 


This study was partly presented as an oral presentation 

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Kiseljaković, Nedjeljka Šljivo, Zakira Mornjaković, 

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Extensive troponin I and T modification detected in 

serum from patiens with acute myocardial infartion. 

Circulation 2000; 102:1221-6. 

17. Nunes JP. Cardiac troponin I in systemic diseases. A 

possible role for myocardial strain. Rev Port Cardiol 

2001; 20:785-8. 

18. Babuin L, Jaffe AS. Troponin: the biomarker of choice 

for the detection of cardiac injury. CMAJ 2005; 

173:119-40. 

19. Dessap AM, Lellouche N, Audard V, Roudot-Thoraval 

F, Champagne S. Effect of renal failure on peak 

troponin I level in patients with acute myocardial 

infarction. Cardiology 2008; 109:217-21. 

20. Wießner R, Hannemann-Pohl K, Ziebig R, Grubitzsch 

H, Hocher B, Vargas-Hein O, Lun A, Schimke 

I, Liefeldt L. Impact of kidney function on plasma 

troponin concentrations after coronary artery 

bypass grafting. Nephro Dial Transplant 2007; 0: 

gfm513v1-gfm513. 

21. O’ Brien PJ, Smith DEC, Knechtel TJ, Marchak MA, 

Pruimboom-Brees, Brees D J , Spratt D P, Archer F 

J, Butler P, Potter AN, Provost J P, Richard J, Snyder 

PA i Reagan WJ. Cardiac troponin I is a sensitive, 

specific biomarker of cardiac injury in laboratory 

animals. Laboratory Animals 2006; 40:153-71. 

Vremenski ovisni odgovori troponina I štakora i oštećenja miokarda nakon administracije 

izoproterenola 

Sabaheta Hasić1 , Radivoj Jadrić1 , Emina Kiseljaković1 , Amina Valjevac1 , Zakira Mornjaković2 , Mira 

Winterhalter-Jadrić1 1 2 Institut za fiziologiju i biohemiju, Institut za histologiju i embriologiju, Medicinski fakultet, Univerzitet u Sarajevu, Bosna i Hercegovina 

SAŽETAK 

Cilj Razviti model infarkta miokarda induciranog aplikacijom izoproterenola (ISO) štakoru. Istraživali 

smo tip histoloških promjena srčanog mišića i kinetiku srčanog troponina I (cTnI). 

Metode Korišteni su odrasli mužjaci Wistar štakori. Štakori su podijeljeni u ISO i kontrolnu grupu. 

Štakori tretirani ISO-om podijeljeni su u grupe prema vremenu analize cTnI i histoloških lezija miokarda: 

ISO I (30’), ISO II (60’), ISO III (120’) i ISO IV (240’). Određivali smo cTnI u serumu ELISA 

metodom (Life Diagnostics Inc., West Chester, PA, USA). Histološku analizu uradili smo na uzorcima 

zida lijevog ventrikula obojenim hematoksilin-eozin metodom bojenja (HE). 

Rezultati Prvo statistički značajno povećanje cTnI zabilježeno je 30 minuta poslije ISO aplikacije. Nije 

utvrđena statistički značajna razlika između ISO grupa u srednjoj vrijednosti cTnI. Histološke promjene 

pokazale su vremensku ovisnost. 

Zaključak Ovaj model, uz određivanje cTnI u krvi, može poslužiti za istraživanje djelovanja kardioprotektivnih 

i kardiotoksičnih supstanci. Sličnost između induciranih lezija miokarda na ovom modelu 

i lezija miokarda čovjeka u ishemiji, dovoljan je podstrek za daljna istraživanja novih kardijalnih markera. 

Ključne riječi: srčani troponin I, izoproterenol, srce, štakor 

22. York M, Scudamore C, Brady S, Chen C, Wilson S, 

Mark Curtis, Gareth Evans, William Griffiths, Matthew 

Whayman, Thomas Williams,John Turton. 

Characterization of troponin responses in isoproterenol-induced 

cardiac injury in the Hanover Wistar 

rat. Toxicol Pathol 2007; 35: 606-61. 

Original submission: 14 October 2010; Revised submission: 08 December 2010; Accepted: 08 December 2010. 

23. 

24. 

25. 

26. 


Bertsch T, Bleuel H, Aufenanger J, Rebel W. Com- 

parison of cardiac troponin T and cardiac troponin I 

concentrations in peripheral blood during orciprenaline 

induced tachycardia in rats. Exp Toxicol Pathol 

1997; 49:467-8. 

Acikel M, Buyukokuroglu ME, Erdogan F, Aksoy 

H, Bozkurt E, Senocak H. Protective effects of dantrolene 

against myocardial injury induced by isoproterenol 

in rats: biochemical and histological findings. 

Int J Cardiol 2005; 98: 389-94. 

Clements P, Brady S, York M, Berridge B, Mikaelian 

I, Nicklaus R, Gandhi M, Roman I, Stamp C, Davies 

D, McGill P, Williams T, Pettit S, Walker D; ILSI 

HESI Cardiac Troponins Working Group, Turton J. 

Time course characterization of serum cardiac troponins, 

heart fatty acid-binding protein, and morphologic 

findings with isoproterenol-induced myocardial 

injury in the rat.Toxicol Pathol 2010; 38:703-14. 

Brooks WW and Conrad CH. Isoproterenol-induced 

myocardial injury and diastolic dysfunction in mice: 

structural and functional correlates. Comp Med 

2009; 59:339–43. 

145

146 


Blood iron stores reduction affects lipoprotein status – a poten- 

tial benefit of blood donation 

Radivoj Jadrić 1 , Sabaheta Hasić 1 , Emina Kiseljaković 1 , Jozo Ćorić 2 , Besim Prnjavorac 3,4 , Mira 

Winterhalter-Jadrić 1 

1 Institute for Physiology and Biochemistry, Medical Faculty, University of Sarajevo, 2 Department for Clinical Chemistry and Biochem- 

istry, Clinical Centre of Sarajevo University; Sarajevo, 3 General Hospital Tešanj, Tešanj, 4 School of Pharmacy, University of Sarajevo, 

Sarajevo; Bosnia and Herzegovina 


Radivoj Jadrić 

Institute for Physiology and Biochemistry 



Čekaluša 90, 71000 Sarajevo 


E-mail: radivoj.jadric@mf.unsa.ba 





Accepted: 


ABSTRACT 


Aim To determine the lipoprotein profile of voluntary blood donors, 

and on the basis of parameters to evaluate the risk of atherosclerosis. 

Methods The study included voluntary blood donors of both 

sexes. Participants were divided into two groups. The first group 

of subjects consisted of men and women in menopause (BD1). The 

second group consisted of women in reproductive age (BD2). Analysis 

of concentration of lipoproteins was performed by direct determination 

of total cholesterol, LDL-C and HDL-C. From the total 

serum cholesterol and concentration of lipoproteins ratios of total 

cholesterol/ HDL-C ratio and LDL-C/HDL-C were calculated. 

Results Significantly higher concentration of LDL-C was obtained 

in the serum of BD 1, compared to LDL-C in the serum of BD 

2, within the reference range. Mean concentration of HDL-C in 

the serum of BD 2 group was higher than the values measured in 

the BD group 1, without significant difference. The ratio of total 

cholesterol / HDL-C showed significantly higher values in the BD 

1 group compared with results in the BD 2 group. Significantly 

higher values in the BD group 1 were observed for the ratio of 

LDL-C/HDL-C. Obtained results showed that all voluntary blood 

donors had a concentration of individual lipoprotein fractions in a 

lower risk range for atherosclerosis development. 

Conclusion Female voluntary blood donors in reproductive age 

have a more favorable lipid status in relation to the voluntary blood 

donors, men and women in menopause, indicating that this 

population of women is exposed to lower risk of developing atherosclerosis. 

Key words: blood donation, iron stores, lipoproteins;

INTRODUCTION 

Although iron deficiency is one of the most common 

nutritional problems existing today around 

the world, iron excess has recently gained attention 

owing to epidemiologic evidence suggesting 

its association with cardiovascular disease, cancer, 

and other diseases. The plausible explanation 

for this association is iron’s pro-oxidant property 

in generating free radicals. Iron is essential in the 

human diet and is needed for many important 

physiologic functions when bound to hemoglobin, 

myoglobin, cytochromes, several enzymes, 

and nonheme iron proteins (1). Results of some 

studies suggest that iron and copper status may 

be associated with lipid peroxidation in subjects 

without metal overload (2) 

Risk factors for atherosclerotic events and cardiovascular 

disease include male sex, increased 

age, elevated plasma total cholesterol (TC) and 

low density lipoprotein cholesterol (LDL-C), 

decreased high-density lipoprotein cholesterol 

(HDL-C), high blood pressure, smoking and 

diabetes mellitus. Approximately 50% of atherosclerotic 

coronary artery disease (CAD) in the 

community occurs in the absence of traditional 

risk factors (3,4) 

The possibility that iron overload plays a role in 

CAD was postulated by J.L. Sullivan in 1981. 

Prominent iron stores may facilitate ischemic illness 

by enhanced reperfusion injury and atherogenic 

properties. The latter mechanism, although 

possibly of outstanding significance, has not yet 

attracted adequate attention in epidemiological 

research. Actually, the post secretory oxidative 

modifications of lipoproteins constitute a crucial 

step in lipid induced atherogenesis, and tissue 

iron may be crucially involved in this process by 

virtue of its outstanding pro-oxidant properties 

(5). Interest in this hypothesis is stimulated by 

its capacity to explain the sex difference in atherosclerotic 

disease and the option of a preventive 

lowering of iron stores by repeated phlebotomy 

(5). In premenopausal women, the incidence of 

atherosclerosis and CAD is less than half of that 

of age-matched men. The female advantage is 

evident in severe hypercholesterolemia, which 

does not affect cardiovascular risk until after menopause 

(5). Depletion of iron stores by regular 

menstrual blood loss may be one source of protection 

in premenopausal subjects (5). Indeed, 

Jadrić et al Blood donation and lipoprotein status 

from a purely mathematical point of view, variation 

of iron stores between sexes could account 

for the sex difference in incident atherosclerosis. 

Iron-deficient men and women constitute a lowrisk 

group, whereas subjects with prominent iron 

stores face a high-risk burden independent of sex 

and menopausal status. Likewise, the gradual 

increase in the incidence of atherosclerosis after 

menopause was best described as a function of 

iron accumulation (5) 

The aim of our prospective study was to determine 

the lipoprotein profile of voluntary blood donors, 

establish influence of additional blood loss by 

menstrual bleeding on lipoprotein profile parameters, 

and to assess the risk of atherosclerosis. 


The reference population consisted of 53 volunteer 

blood donors from both sexes aged from 30 

to 55 years attending Sarajevo Blood Transfusion 

Center during the period of one year. The ethical 

committee of our institution approved the trial. 

Study participants were divided in two groups. 

Male participants with women in menopause 

were classified in group one (n=37, BD1), while 

group two consisted of women in reproductive 

age, with additional iron loss by menstrual bleeding 

(n=16, BD2). 

Venous blood samples were taken and centrifuged, 

sera separated, transferred to a fresh tube for 

determination of lipid parameters, and stored at 

-20 °C until analyses were performed. Analyses 

were performed by measuring total cholesterol, 

and direct measuring of LDL-C and HDL-C. Indexes 

of atherosclerosis were calculated from total 

cholesterol/HDL-C and LDL-C/HDL-C ratio. 

Statistical analysis was performed by using 

Student’s T test, and results given as mean value ± 

standard error of the mean. We considered p value 

less than 0.05 (p

148 


Figure 1. Total cholesterol concentration in blood donor sera* 

*BD 1, male participants with women in menopause (n=37); 

BD 2, women in reproductive age (n=16) 

nors (BD 1) were significantly higher than the 

value in the serum of women in the reproductive 

age (BD 2) (p

Figure 5. Values of LDL/HDL ratio* 

*BD 1 - male participants with women in menopause (n=37); 

BD 2 - women in reproductive age (n=16) 

loss by menstrual bleeding, and thus reduced 

oxidation of cholesterol, is likely contribute to a 

lesser tendency to the development of atherosclerosis 

in blood donors with the additional loss of 

iron with menstrual blood. Data on presumably 

lower serum iron stores concentration of women 

in the reproductive age, in which concentrations 

of lipids, lipoproteins, and the relationship of total 

cholesterol/HDL and LDL/HDL is reduced speak 

in favor of the hypothesis that the decreased concentration 

of iron has an impact on reducing the 

concentration of lipids and lipoproteins. Risks of 

occurrence of cardiovascular diseases, according 

to previous studies are associated with the status 

of iron metabolism (8). 

Our study findings are supported by investigations 

of Baradhway (9). As it was shown in our 

study, blood donor lipid profiles were in the 

normal range. The blood donors appear to be a 

protected group with a reduced risk of CAD as 

has been substantial by various past studies (10, 

11). The reason for this reduction was attributed 

to the “iron hypothesis” which proposed that the 

reduction in the iron load with each blood dona- 

REFERENCES 

1. 

2. 

3. 

Mehrabani M, Djalali M, Sadeghi MR, Hajibeigi 

B, Zeraati H, Fatehi F, Chamari M. Association 

between blood donation frequency, antioxidant enzymes 

and lipid peroxidation. Acta Medica Iranica 

2008; 46:361-6. 

Arnaud J, Fleites P, Chassagne M, Verdura T, Renversez 

J-C, Garcia Garcia I, Tressol JC, Favier AE, 

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between serum lipoperoxide concentrations and iron 

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Georgiadis AN, Papavasiliou EC, Lourida ES, Alamanos 

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lipid profile is a feature characteristic of patients 

with early rheumatoid arthritis: effect of early 

treatment – a prospective, controlled study. Arthritis 

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ar1952). 

Jadrić et al Blood donation and lipoprotein status 

tion was the reason for the fall in incidence of 

cardiovascular problems. This has since been disproved 

by other studies (7). Further studies have 

borne out that blood donation may be beneficial 

by increasing the serum HDL concentration (6), 

which shows optimal values in all of our study 

subjects. Also HDL concentration may be considered 

a treatment modality for hypercholesterolemia 

(12). The Meyers study revealed that the 

benefit of blood donation was limited to the male 

donors who had donated blood in the most recent 

last 3 years (13, 9). Findings of Mehrabani et al. 

(1) is consistent with the hypothesis that reduction 

in iron stores is associated with decreased 

oxidative stress. Results from a study in patients 

with coronary artery disease, showed an association 

of higher serum ferritin with increased lipid 

peroxidation (14). 

More research is needed in future to help better 

understanding of the nature of this association 

before appropriate actions can be developed. Dietary 

intakes do not explain the difference in the 

prevalence of iron deficiency between women of 

different ethnic origin, which suggests that the 

etiology of iron deficiency may depend on other 

factors. Prospective studies, especially intervention 

studies that ensure adequate iron status, are 

needed to compare 2 possible scenarios, improved 

iron status increases the risk of CAD, or patients 

with a higher risk of CAD have higher iron 

stores, with both factors resulting from the same 

underlying cause (15, 8). 



4. Cui Y, Blumenthal RS, Flaws JA, Whiteman MK, 

Langenberg P, Bachorik PS, Bush TL. Non-highdensity 

lipoprotein cholesterol level as a predictor 

of cardiovascular disease mortality. Arch Intern Med 

2001; 161:1413-9. 

5. Kiechl S, Willeit J, Egger G, Poewe W, Oberhollenzer 

F. Body iron stores and the risk of carotid atherosclerosis 

circulation. 1997; 96:3300-7. 

6. van Jaarsveld 

H, Pool GF. Beneficial effects of blood 

donation on high density lipoprotein concentration 

and the oxidative potential of low density lipoprotein. 

Atherosclerosis 2002; 161:395-402 

7. Ascherio A, Rimm EB, Giovannucci E, Willett WC, 

Stampfer MJ. Blood donations and risk of coronary 

heart disease in men. Circulation 2001; 103:52-7. 

149

150 


8. Ramakrishnan U, Kuklina E, Stein AD. Iron stores 

and cardiovascular disease risk factors in women 

of reproductive age in the United States. Am J Clin 

Nutr 2002; 76:1256–60. 

9. Bharadwaj RS. A study of lipid profiles among male 

voluntary blood donors in Chennai city. Indian J Community 

Med 2005; 30:16-17 

10. Pekka T, Salonen R. Cohort study of relationship 

between donating blood and the risk of myocardial 

reaction. BMJ 1997; 314:793-4. 

11. Salonen JT, Toumainen TP. Donating blood is associated 

with reduced risk of myocardial infarction. 

The Kuopio ischemic heart disease risk factor study. 

Am J epidemiol 1998; 148:445-51. 

12. Kumar H. Repeated blood donations effective in 

treating hyperlipidemia. J Assoc Physicians India. 

1994; 42:468-9. 

Smanjivanje zaliha željeza u krvi utiče na status lipoproteina – potencijalna korist od 

darivanja krvi 

Radivoj Jadrić1 , Sabaheta Hasić1 , Emina Kiseljaković1 , Jozo Ćorić2 , Besim Prnjavorac3,4 , Mira 

Winterhalter-Jadrić1 1 2 Institut za fiziologiju i biokemiju, Medicinski fakultet Univerziteta u Sarajevu, Odjel za kliničku kemiju i biokemiju, Klinički centar Univerziteta 

u Sarajevu; Sarajevo; 3Opća bolnica Tešanj, Tešanj; 4Farmaceutski fakultet Univerziteta u Sarajevu, Sarajevo; Bosna i Hercegovina 

SAŽETAK 

Cilj Odrediti lipoproteinski profil dobrovoljnih darivatelja krvi, te na temelju ovih parametara procijeniti 

rizik od nastanka ateroskleroze. 

Metode U istraživanju su sudjelovali dobrovoljni darivatelji krvi oba spola. Ispitanici su bili podijeljeni 

u dvije skupine. Prvu grupu ispitanika činili su dobrovoljni darivatelji krvi muškog spola i žene u menopauzi 

(BD 1), dok se druga grupa sastojala od dobrovoljnih darivatelja krvi, žena u reproduktivnoj dobi 

(BD 2). Analiza koncentracije lipoproteina izvršena je direktnim određivanjem ukupnog kolesterola, 

LDL-C i HDL-C. Na osnovu koncentracije ukupnog kolesterola u serumu i koncentracije lipoproteina 

izračunati su odnosi ukupnog kolesterola i HDL-C, te odnos LDL-C/HDL-C. 

Rezultati Ustanovljena je statistički značajno veća koncentracija LDL-C u serumu grupe BD 1 u odnosu 

na LDL-C u serumu grupe BD 2, ali unutar referentnog raspona. U isto vrijeme, srednja koncentracija 

HDL-C u serumu grupe BD 2 bila je viša od vrijednosti izmjerene u grupi BD 1, ali bez statistički 

značajne razlike. Omjer ukupnog kolesterola/HDL-C imao je značajno veće vrijednosti u grupi BD 1 u 

usporedbi s istim omjerom određenim u drugoj skupini, BD 2. Značajno veće vrijednosti u grupi BD 1 

uočene su za omjer LDL-C/HDL-C. Dobiveni rezultati pokazali su da svi dobrovoljni darivatelji krvi 

imaju koncentraciju pojedinih lipoproteinskih frakcija u području nižeg rizika za razvoj ateroskleroze. 

Zaključak Dobrovoljni darivatelji krvi ženskog spola u reproduktivnim godinama, imaju povoljniji 

status metabolizma lipida u odnosu na dobrovoljne darivatelje krvi muškog spola i žene u menopauzi. 

Ove činjenice ukazuju da je ova populacija žena izložena manjem riziku od razvoja ateroskleroze. 

Ključne riječi: darivanje krvi, depoi željeza, lipoproteini. 

13. Meyers DG, Strikland D. Possible association of a 

reduction in cardiovascular events with blood donation. 

Journal of heart 1997; 78:188-93. 

14. Yesilbursa D, Serdar Z, Serdar A, Dirican M, Gemici 

K, Özdemir A, Türel B, Cordan J. The relationship 

of serum ferritin with malondialdehyde concentration 

in patients with coronary artery disease: Feritin 

and oxidative stress in CAD. Int J Angiol 2001; 

10:88-91. 

15. Frith-Terhune A, Cogswell M, Kettel-Khan L, Will 

J, Ramakrishnan U. Determinants of iron deficiency 

among Mexican-American and non-Hispanic white 

females: third National Health and Nutrition Examination 

Survey, 1988-94 (NHANES III). Am J Clin 

Nutr 2000; 74:963-8. 



Hematologic and laboratory parameters in patientis with peptic 

ulcer bleeding treated by two modalities of endoscopic haemostasis 

and proton pompe inhibitors 

Amila Mehmedović-Redžepović 1 , Rusmir Mesihović 1 , Besim Prnjavorac 2,4 , Aida Kulo 3 , Kalajdžija Merlina 5 

1 2 Department of Gastroenterology and Hepatology, Refferal Centre for Gastrointestinal Endoscopy, Clinical Centre, Department of Patophysiology, 

School of Pharmacy, 3Institute of Pharmacology, Clinical Pharmacology and Toxicology, School of Medicine; University of 

Sarajevo, 4 General Hospital Tesanj, 5Department of Anesthesiology, Cantonal Hospital Zenica, Zenica; Bosnia and Herzegovina 


Amila Mehmedović-Redžepović 

Department of Gastroenterology and 

Hepatology, Refferal Centre for Gastrointestinal 

Endoscopy, Clinical Centre, 

University of Sarajevo, 

Bolnička 25, 71000 Sarajevo, 


Phone: +387 33 220 132 

E-mail: amila.redzepovic@gmail.com 





Accepted: 


ABSTRACT 


Aim To compare two schedules (continuous infusion or bolus iv. of 

PPI) in treatment after endoscopic homeostasis of bleeding ulcers. 

Methods Patients with gastrointestinal bleeding caused by peptic 

ulcer, or a recent history (

152 


INTRODUCTION 

The management of peptic ulcer bleeding has been 

revolutionized in the past two decades with the advent 

of effective endoscopic hemostasis and potent 

acid-suppressing agents (PPI). It is best managed 

using a multidisciplinary approach by the team 

with medical, endoscopic and surgical expertise. A 

prompt initial clinical and endoscopic assessment 

should allow patients to be triaged effectively into 

those who require active therapy, versus those 

who require monitoring and preventative therapy. 

A combination of pharmacologic and endoscopic 

therapy (using a combination of injection 

and thermal coagulation) offers the best chance of 

hemostasis for those with active bleeding ulcers. 

Surgery, being the most effective way to control 

bleeding, should be considered for conservative 

treatment failures. The choice between surgery 

and repeat endoscopic therapy should be based on 

the pre-existing comorbidities of the patient and 

the characteristics of the ulcer. (1) 

In patients with upper gastrointestinal bleeding 

caused by peptic ulcer disease, intravenous proton-pump-inhibitor 

therapy seems to be beneficial. 

Following successful endoscopic hemostasis, 

Lau et al. (2) demonstrated that the use of 

intravenous IPP 40 mg every 12 h reduced the 

rebleeding rate from 22.5% to 6.7%, this increase 

in frequency seemed to decrease the volume of 

blood transfusion needed. 

After 3 days of intravenous IPP treatment, the patient 

can be switched to daily 40 mg by mouth for 

8 weeks (2). Therapy can be stopped after 8 weeks 

unless the patient has an associated Helicobacter 

pylori infection, is maintained on low-dose aspirin, 

or uses a nonselective NSAID. Patients with 

H. pylori infection are at higher risk of rebleeding 

and should receive a 14-day course of antibiotics 

in addition to proton-pump inhibitors (2,3). 

Patients taking low-dose aspirin or nonselective 

NSAIDs are at high risk from the development of 

recurrent ulcers and should receive proton-pumpinhibitor 

maintenance therapy (4,5). Patients who 

are H. pylori-negative, NSAID-negative, and have 

an idiopathic bleeding ulcer may not require any 

further therapy after the 8-week course of therapy 

is completed. Clearly, the best approach to this 

group of patients has yet to be determined. 

For patients with bleeding peptic ulcers that display 

major endoscopic stigmata of recent hemorr- 

hage, a combination of endoscopic and pharmacologic 

therapy is the current standard management; 

however, the optimal regimen for administration 

of proton pump inhibitors (PPIs) remains controversial. 

Two consensus documents have endorsed 

a high-dose PPI regimen (80 mg stat followed by 

an infusion of 8 mg/h for 72 h (6,7). The biologically 

plausible mechanism of benefit of such a 

high-dose regimen is to promote clot stability by 

sustaining the intragastric pH above 6 (8,9). Once 

primary hemostasis is achieved by endoscopic 

therapy, clinical trials show that a high-dose PPI 

infusion is superior to placebo (10,11); however, 

when the comparator is a standard-dose PPI regimen, 

four prospective trials (12-15) and a metaanalysis 

(16) report no difference in the magnitude 

of risk reduction between the intensive- and 

the low-dose regimens. 

We conducted a study, comparing two strategies 

for intravenous PPI administration in the prevention 

of rebleeding, surgery, and death in patients 

with high-risk bleeding peptic ulcers in whom 

successful endoscopic hemostasis was achieved. 

The metabolism of PPIs is dependent upon P450 

2C19 genotypes and the clinical usefulness of genotypic 

analysis remains to be determined. 


A randomized, study of PPI therapy for the prevention 

of ulcer rebleeding in high-risk patients 

after endoscopic hemostasis was conducted by 

well experienced team of the Clinical Centre Department 

of Gastroenterology and Hepatology in 

the period between January and December 2008. 

Eligible patients were those with gastrointestinal 

bleeding or recent history (less than 24 hrs before 

admission) of hematemesis and/or melena in our 

Hospital Emergency Departments, as were the patients 

whose ulcer hemorrhage started after the hospitalization 

for an unrelated medical or surgical condition. 

The reference time was the onset of symptoms 

and signs, or when the bleeding started, if the patient 

was already in hospital for other reasons. 

Eligible patients were had to have an ulcer with 

either active bleeding (spurting arterial or persistent 

oozing) or a nonbleeding lesion (nonbleeding 

visible vessel or adherent clot) at endoscopy. 

According to Rockall’s criteria, (17) patients determined 

to be at high clinical risk for rebleeding 

clinically were those with a Rockall score ≥6,

calculated on patients’ demographic and clinical 

characteristics or volume and rapidity of blood 

loss: age ≥70 year; concomitant illness, defined as 

a medical history of chronic illness or presence of 

acute medical condition; transfusion of ≥2 units 

of packed blood cells or hemoglobin ≤100 g/L; 

and hemodynamic instability, defined either by 

hypotension (systolic blood pressure ≤90 mmHg) 

or tachycardia (heart rate ≥100 beats per min). 

Hemodynamically unstable patients were initially 

resuscitated and then considered for enrolment if 

their condition stabilized. Exclusion criteria were 

malignant-appearing ulcers, severe comorbid 

conditions, oesophageal varices, severe coagulopathy 

or platelet count

154 


shown between the groups with respect to pantoprazol 

or omeprazol medication. All patients 

completed the assigned schedule of IPPs administration. 

No drug-related side effects were reported 

in either group. Mean number of units of 

blood transfusion for patients in the intensive 

group was 2,18 (SD 0,8) and 1,34 (SD 1,02) in 

the standard group, with statistical level of difference 

p = 0.0004, using Student t-test. 

Bleeding recurred in 5 patients (14.7%) in the 

intensive regimen group and in 8 (22.8%) in the 

standard regimen group. At the time on admission 

to hospital, hemoglobine rate in the standard 

regimen group was 93,5 g/l (SD 23,8), and in 

the intensive regimen group it was 106,6 g/l (SD 

22,4) ( p = 0.042). At the repeat endoscopy, 0 

lesions were spurting ulcers, 7 oozing, 5 had a 

Table 1. Characteristics of patients included in the analysis* 

Characteristic 

Intensive regimen 

(N = 34) 

Standard regimen 

(N = 35) 

Age–yr 55.0 ( SD 16.9) 62.3 ( SD 17,9) 

Age > 70 yr–no. (%) 7 (20.5) 15 (42.8) 

Men–no. (%)-Women-no(%) 21 (67)-13(33) 

24 (68,5)-11 

(31,5) 

Hemoglobin–g/L 

Patients with hemoglobin 

106.6 ( SD22.4) 

95.5 (SD 23.8) 

P = 0.042102 

≤ 100 g/l–no. (%) 14 (41.1) 19 (54.3) 

Shock at presentation–no. (%) 

Rockall score 

3(8.8) 5 (14.2) 

Mean ± SD 6.2 ( 1.2) 6.6 ( 0.9) 

≥6 points–no. (%) 25 (73.5) 29 (82.8) 

Severe comorbidity–no. (%) 

Bleeding during hospitalization 

3 (8.8) 5 (14.2) 

no. (%) 3 (8.8) 4 (11.4) 

Risk factors for bleeding peptic ulcer–no. (%) 

Use of NSAID 12 (35.3) 14 (40) 

Use of aspirin 11 (32.4) 12 (34.2) 

Time from bleeding to endoscopy 

≤6 h 7 (20.5) 8 (22.8) 

Between 6 and 12 h 9 (26.5) 9 (25.7) 

Between 12 and 24 h 14 (41.2) 12 (34.3) 

>24 h 4 (11.8) 6 (17.2) 

Previous ulcer disease–no. (%) 19 (55.8) 15 (42.8) 

Previous bleeding–no. (%) 8 (23.5) 4 (11.4) 

Peptic ulcer as source of bleeding–no. (%) 

Duodenal ulcer 10 (29.5) 8 (22.8) 

Gastric ulcer 

Forrest classification–no. (%) 

24 (70.5) 27 (77.2) 

1 a 6 (17.6) 5 (14.3) 

1 b 11 (32.4) 13 (37.1) 

2 a 12 (35.3) 15 (42.9) 

2 b 5 (14.7) 2 (5.7) 

Endoscopic hemostasis modality–no. (%) 

Unimodal 18 (52.9) 20 (57.1) 

Multimodal 16 (47.1) 15 (42.9) 

Type of PPI administered–no. (%) 

Omeprazole 5 (14.7) 3 (8.6) 

Pantoprazole 29 (85.2) 32 (91.4) 

*NSAID, nonsteroidal anti-inflammatory drug; PPI, proton pump 

inhibitor; SD, standard deviation 

nonbleeding visible vessel, 1 an adherent clot. 

Between the two treatment groups, rebleeding 

rates did not differ for gastric and duodenal ulcers. 

There were no differences in rebleeding 

rates between the two treatment groups for each 

of the endoscopic stigmata of recent hemorrhage, 

for patients who received endoscopic monotherapy 

versus those who underwent multimodal intervention, 

and for patients with baseline Rockall 

score >6 versus 24 h to 7 days 3 (8.8) 3 (8.6) 

Rebleeding according to Rockall score: no. (%) 

Mean globulin rate in the standard regimen group 

was 31,0 g/l (SD 5,2) and 32,3 g/l (SD 5,3) (p = 

0.875) in the intensive regimen group. Mean fibrinogen 

rate in the standard regimen group was 

11,1 mmol/l (SD 2,6) and 10.8 mmol/l (SD 2,4) 

(p = 0.622) in the intensive regimen group. 

One patient died during the hospital stay; the cause 

of death was not linked to the bleeding event . 

DISSCUSION 

In patients with ulcer bleeding which was successfully 

treated by endoscopy, we found no 

evidence that an intensive dose PPI regimen reduced 

rebleeding compared with a less-intensive 

regimen of bolus injections: bleeding recurred 

in 14.7% and 22.8% of patients, respectively. 

The statistically equal rebleeding rate between 

the two intravenous IPP strategies is of relevance 

considering that patients treated with bolus 

IPPs more frequently had shock and signs of hemodynamic 

instability, two predictors of worse 

outcome (16,18). 

However, in patients who underwent simultaneous 

endoscopic hemostasis, two trials reported 

opposite results with the intensive PPI dosing 

versus placebo (12, 19) a single report showed 

a benefit of high-dose PPI therapy in comparison 

with H2R antagonists (20), and four studies 

did not show a significant difference between an 

intensive regimen and standard bolus PPI injections 

(13-16). 

As intravenous PPI therapy is expensive, in order 

to be cost-effective an extra cost of the medication 

must be compensated by a reduction in the occurrence 

of important adverse clinical outcomes. 

Theoretically, outcome of treatment depends 

on applied modality of endoscopic hemostasis, 

whether the epinephrine injection (1:10,000 dilution 

in saline, 1–1,5 ccm/injection) was administered 

as monotherapy (unimodal) or in association 

with either thermal or mechanical therapy 

(multimodal). 

Pharmacologic data indicate a class effect of PPI 

therapy as inhibitors of gastric secretion; however, 

Mehmedović-Redžepović et al Laboratory parameters in bleeding ulcer 

it is not always clear whether subtle variations in 

the pharmacokinetics and pharmacodynamics of 

individual PPIs are necessarily of clinical importance. 

Indeed, a similar rate of rebleeding has been 

found in a head-to-head comparative trial of omeprazole 

versus pantoprazole (21). As for the claim 

of a better outcome after dual endoscopic therapies 

as opposed to monotherapy with epinephrine injection 

(22,23), published data are not consistent.. We 

add to the ongoing debate our finding of similar 

rebleeding rates after hemostasis being achieved 

with either mono- or dual-endoscopic treatment. 

Moreover, cost-effectiveness analyses have yielded 

contrasting results regarding which one 

of the two PPI administration strategies is more 

effective in patients who have received concomitant 

endoscopic treatment. Furthermore, it has 

been shown that H. pylori-infected patients may 

respond differently to PPIs therapy than noninfected 

patients (24), but this information could 

be retrieved only for a third of enrolled patients 

in our study, therefore precluding the evaluation 

of its impact on the therapeutic outcomes of the 

study. 

In this study, patients who received the standard 

PPI regimen had advantage with respect to transfusion 

requirement, but no advantage as for the 

need for surgery, length of hospital stay, or death 

rate. A final shortcoming might be the reporting 

only for in-hospital events and exclusion of the 

data on 30-day outcomes. Considering the mean 

duration of hospital stay of our patients, we feel 

confident that the number of missed negative outcomes 

was very low. 

In patients with bleeding peptic ulcers with successful 

endoscopic hemostasis the standard PPI 

regimen had advantage of transfusion requirements, 

but no advantage with respect to in-hospital 

rates of rebleeding rates, need for surgery, 

length of hospital stay, or death, which is in 

accordance with recent studies (11,25). 



155

156 


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Sung JJ, Lee KK, Yung MY, Wong SK, Wu 

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H, Boesby S, Snel P, Vreeburg E.M, Eriksson 

S, Fernstrom P, Hasselgren H. Effects of omeprazole 

on the outcome of endoscopically treated peptic ulcers. 

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11. 11.Andriulli A, Loperfido S, Focareta R, Leo P, Fornari 

F, Garripoli A, Tonti P, Peyre S, Spadaccini A, 

Marmo R, Merla A, Caroli A, Battista Forte G, Belmonte 

A, Aragona G, Imperiali G, Forte F, Monica 

F, Caruso N, Perri F. High- Versus Low-Dose Proton 

Pump Inhibitors After Endoscopic Hemostasis in 

Patients With Peptic Ulcer Bleeding: A multicentre, 

randomized study. Am J of Gastroenterol 2008; 

103:3011-8. 

12. Schonekas H, Ahrens H, Pannewick U, Ell C, Koop 

H, Petrisch W, Klein M, Fischer R. Comparison of 

two doses of intravenous pantoprazole in peptic ulcer 

bleeding. J Gastroenterol 1999; 116:A305. 

13. Udd M, Miettinen P, Palmu A, Heikkinen M, Janatuinen 

E, Pasanen P, Tarvainen R, Kairaluoma 

MV,Lohman M, Mustonen H, Julkunen R. Regulardose 

versus high-dose omeprazole in peptic ulcer 

bleeding: A prospective randomized double-blind 

study. Scand J Gastroenterol 2001;36:1332–8. 

14. Dokas SM, Lazaraki GI, Kontoninas Z, Kouklakis 

GS, Adamidou A, Tsiaousi E, Christoforidis C, 

Ziakas G.Bolus intravenous omeprazole b.i.d vs. 

continuous intravenous omeprazole infusion combined 

with endoscopic hemostasis in the treatment of 

peptic ulcer bleeding. Preliminary results. Gut 2004; 

53(Suppl VI):A290. 

15. Bajaj JS, Dua KS , Hanson K, Presberg K.Prospective, 

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upper gastrointestinal bleeding: A pilot study. 

Dig Dis Sci 2007; 52:2190–4. 

16. Andriulli A, Annese V, Caruso N, Pilotto A, Accadia 

L, Niro AG, Quitadamo M, Merla A, Fiorella S, 

Leandro G.Proton-pump inhibitors and outcome of 

endoscopic hemostasis in bleeding peptic ulcer: A 

series of meta-analysis. Am J Gastroenterol 2005; 

100:207–19. 

17. Rockall TA, Logan RF, Devlin HB, Northfield TC. 

Risk assessment after acute upper gastrointestinal 

haemorrhage. Gut 1996; 38:316–21. 

18. Lin HJ, Lo WC, Chen YC, Perng CL. Role of intravenous 

omeprazole in patients with high-risk of peptic 

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trial. Am J Gastroenterol 2006; 101:500–5. 

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20. Fried R, Beglinger C, Meier R, Stumpf J, Alder G, 

Schepp W. Comparison of intravenous pantoprazole 

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21. Chilovi F, Piazzi L, Zancanella L, De Guelmi A, 

Grasso T, Di Fede F, Bertozzo A, Amplatz S, Farris 

P, Benvenuti S. Intravenous omeprazole and pantoprazole 

after endoscopic treatment of bleeding peptic 

ulcers. Gastrointest Endosc 2003; 57:AB150. 

22. Marmo R, Rotondano G, Piscopo R, Bianco MA, 

D’Angella R, Cipolletta L. Dual therapy versus monotherapy 

in the endoscopic treatment of high-risk 

bleeding ulcers: A meta-analysis of controlled trial. 

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23. ASGE guidelines: The role of endoscopy in acute 

non-variceal upper-GI hemorrhage. Gastrointest Endosc 

2004; 60:497–504. 

24. Pilotto A, Franceschi M, Longoa MG, Scarcelli C, 

Orsitto G, Perri FC. Helicobacter pylori infection 

and the prevention of peptic ulcer with proton pump 

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Dig Liver Dis 2004; 36:666–70. 

25. Jensen DM, Pace SC, Soffer E, Comer GM, Members 

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study. Am J Gastroenterol 2006; 101:1991–9.

Praćenje hematoloških i laboratorijskih parametara kod krvarećeg ulkusa primjenom 

dva protokola hemostaze, endoskopijom i inhibitorima protonske pumpe 

Amila Mehmedović-Redžepović1 , Rusmir Mesihović1 , Besim Prnjavorac2,4 , Aida Kulo3 , Kalajdžija Merlina5 

1Klinika za gastroenterologiju i hepatologiju, Referalni centar za gastrointestinalnu endoskopiju, Klinički centar Univerziteta u Sarajevu, 

2 3 Katedra za patofiziologiju, Farmaceutski fakultet, Institut za farmakologiju, kliničku farmakologiju i toksikologiju, Medicinski fakultet; 

Univerzitet u Sarajevu; 4Opća bolnica Tešanj; 5Odjel za anesteziologiju, Kantonalna bolnica Zenica; Bosna i Hercegovina 

SAŽETAK 

Mehmedović-Redžepović et al Laboratory parameters in bleeding ulcer 

Cilj Usporedba dva režima primjene IPP-a (kontinuirana infuzija ili i.v.) pri tretmanu gastrointestinalne 

hemoragije, nakon endoskopske intervencije. 

Metode Ispitanici s kliničkom slikom aktivne ili tek nedavno, unutar 24 sata, zaustavljene gastrointestinalne 

hemoragije uzrokovane ulkusnom bolešću, ili s pozitivnom anamnezom gastrointestinalne 

hemoragije. Pacijenti su randomizirani u dvije grupe. Jedna je grupa primala intravenozno IPP u kontinuiranoj 

infuziji (5 amp 40 mg u kontinuiranoj infuziji 8 mg/h u 72 h) – intenzivni režim; a druga je 

grupa primala IPP po standardnom režimu (40 mg amp PPI dva puta dnevno, u trajanju od tri dana), a 

potom peroralno IPP a 40 mg dva puta dnevno. 

Rezultati Tretirano je ukupno 69 pacijenata. Do ponovnog krvarenja došlo je kod 5 od 34 pacijenta 

(14,7%) na intenzivnom režimu, te kod 8 od 35 (22,8%) pacijenata na standardnom režimu. Nivo hemoglobina 

u grupi na standardnom režimu bio je 93,5 g/l (SD 23,8), a na intenzivnom režimu 106,6 g/l 

(SD 22,4) (p= 0,042). Prosječna plazmatska razina ukupnih proteina kod pacijenata standardne grupe 

bila je 65,1 g/l (SD 7,3), a u grupi intenzivnog režima 67,7 g/L (SD 8,15) (p= 0,525). Nivo albumina 

u standardnoj grupi bio je prosječno 31,0 g/L (SD 5,2), a u intenzivnoj 34,8 (SD 7,4) (p= 0,652). Nivo 

globulina u standardnoj grupi bio je prosječno 31,0 g/L ( SD 5,2), a u intenzivnoj 32,3 g/L (SD 5,3) (p= 

0,875). Fibrinogen u standardnoj grupi iznosio je 11,1 mmol/l (SD 2,6), a u intenzivnoj 10,8 mmol/L 

(SD 2,4) (p= 0,622). Prosječan broj potrebnih transfuzija krvnih derivata za pacijente grupe intenzivnog 

režima bio je 2,18 (SD 0,8), odnosno 1,34 (SD 1,02) za grupu sa standardnim režimom (p= 0,0004). 

Prosječan broj dana hospitalizacije bio je 6,4 (SD 2,8) za grupu standarnog i 5,8 (SD 2,8) za grupu 

intenzivnog režima (p= 0,40). Hirurški tretman bio je potreban kod 2 pacijenta na intenzivnom i kod 

jednog na standardnom protokolu. 

Zaključak Kod pacijenata s gastrointestinalnom hemoragijom kod ulkusne bolesti, nakon endoskopske 

hemostaze, standardni režim ordiniranja IPP-a imao je prednosti nad intenzivnim u potrebi za transfuzijom 

krvi, ali nije imao prednosti u smanjenju ponovnih krvarenja, potreba za hirurškom intervencijom, 

dužine potrebne hospitalizacije ili pojave smrtnog ishoda. 

Ključne riječi: krvareći ulkus, inhibitori protonske pumpe, endoskopska hemostaza 

Original submission: 01 November 2010; Revised submission: 05 December 2010; Accepted: 07 December 2010. 

157

158 


Evaluation and treatment of cardiovascular diseases in patients 

on hemodialysis – single center experience 

Halima Resić 1 , Besim Prnjavorac 2 , Fahrudin Mašnić 1 , Selma Ajanović 1 , Nihad Kukavica 1 , Amela 

Bećiragić 1 

1 2 Clinic for Hemodialysis, Clinical Center University of Sarajevo, Sarajevo, Center for Hemodialysis, General Hospital Tešanj, Tešanj; 



Resić Halima 

Clinic for Hemodialysis, Clinical Center 



Phone/fax: +387 33 269 071 

E-mail: kcushemodijaliza@bih.net.ba 





Accepted: 


ABSTRACT 


Aim To evaluate frequency of CVD in dialysis population, in relationship 

to patients with and without diabetes, and their most 

common treatment. 

Patients and methods This retrospective study included 187 patients, 

106 males and 81 females, divided in two groups, diabetics 

and non-diabetics, treated by chronic hemodialysis. Patients’ 

analyses included: anamnesis, ECG, chest X-rays, echocardiogram, 

laboratory examinations for calcium (Ca), phosphorus (P), 

parathormone (PTH), cholesterol (chol), triglicerids (TG), C-reactive 

protein (CRP), hemoglobin (Hb) and uric acid. In addition, 

we analyzed groups of drugs used by patients as prescribed by 

cardiologists. 

Results Average age was 58.0 years, most of them between 51 

and 60. Average hemodialysis length was 4 years. Primary kidney 

diseases were pyelonephritis and glomerulonephritis. 19,78% of 

patients had diabetes. 165 patients (88,23%) had one or more cardiovascular 

diseases. 110 patients (58,2%) had hypertension, most 

of them used ACE inhibitors. Using test of multiple correlation, 

statistically significant correlations, among others, were shown 

between BMI and Ca, uric acid and P, albumin and PTH in diabetics, 

at the statistical significance level at p

INTRODUCTION 

Cardiovascular disease (CVD) is prevalent in patients 

receiving dialysis therapies. It represents 

major cause of morbidity and mortality in patients 

with end-stage renal disease (ESRD) (1). It affects 

long-term hemodialysis outcomes. It is important 

to evaluate the extent of all aspects of CVD in dialysis 

patients. In those patients with limited life 

expectancy due to severe non-cardiac comorbidity, 

evaluation and therapy should be individualized. 

Risk factors for CVD in patients with ESRD can 

be divided into those specific to ESRD and those 

that are nonspecific to kidney disease but are more 

prevalent. Many traditional risk factors for CVD 

(diabetes, dyslipidemia, hypertension, age, male, 

gender, physical inactivity) are becoming quite 

high (2). Patients with ESRD also have additional 

risk factors such as anemia, hyperparathyroidism, 

oxidative stress, chronic inflammation, hypoalbuminemia, 

hyperhomocysteinemia, prothrombotic 

factors. Hemodialysis treatment itself may also 

contribute to CVD (2). Data suggest that uremic 

factors, or factors related to renal replacement 

therapy (RRT)/dialysis may be implicated in the 

pathogenesis of heart disease in patients treated by 

dialysis, because cardiovascular survival improves 

after transplantation even in high-risk patients (3). 

Appearance of CVD is very common in dialysis 

patients and accounts for almost 50% of deaths 

(4). Since only a small fraction of patients with 

chronic kidney disease (CKD) progress to ESRD 

and requires RRT or renal transplantation, focus 

needs to be shifted to the development of CVD. 

The aim of the study was to evaluate frequency of 

CVD in our dialysis population in relation to patients 

with and without diabetes and to analyze type 

of treatment for different cardiovascular diseases. 


This retrospective study included 187 patients on 

chronic hemodialysis program at the Clinic for 

Table 1. Distribution of patients by age group 

Age group No of patients (%) 

81 2 (1.1%) 

Total 187 (100.1%) 

Resić et al Cardiovascular diseases and hemodialysis 

Hemodialysis, Clinical Center of the University 

of Sarajevo. Patients were evaluated and treated 

for CVD. The main criterion for patient inclusion 

in the study was the duration of hemodialysis 

for at least 6 months. Patients were dialyzed 3 

times weekly for 4 hours on the Fresenius machines 

4008S, 4008B, with bicarbonate dialysis. 

CVD diagnostic techniques were the following: 

anamnesis, ECG, chest and heart x-rays, heart 

ultrasound, dry weight assessment. The criteria 

for CVD diagnosis on the basis of ECG were as 

follows: ventricle extrasystoles and ventricle tachycardia, 

sinus tachycardia and specific ST segment 

changes in dilating myocardiopathia, ST 

segment depressions and deep S waves in V1-V3 

leads in hypertrophic myocardiopathia and also, 

QRS complex changes. The criteria for CVD diagnosis 

using heart ultrasound were as follows: 

typical enlargement of interventricular septum 

with turbulence at the exit part of the ventricle in 

hypertrophic myocardiopathia and, also enlargement 

of all atria and ventricles in dilating myocardiopathia. 

In the research of individual cardiovascular 

diseases we analyzed groups of drugs 

used by patients as prescribed therapy. On the basis 

of analysis of used pharmacotherapy we tried 

to determine which group of drugs gives better 

effects in the treatment. Laboratory examinations 

for calcium (Ca), phosphorus (P), parathormone 

(PTH), cholesterol (chol), triglycerides (TG), Creactive 

protein (CRP), hemoglobin (Hb) and uric 

acid were done accordingly. Descriptive statistics 

was used for mean value, standard deviation and 

test of multiple correlations. A comparison of laboratory 

parameters between diabetics and non 

diabetics was performed using Student t-test. 

RESULTS 

Our study involved 187 patients, 106 males 

(56.68%) and 81 females (43.32%), mean age 58 

years (SD 13.69) (Table 1). Dialysis length was 

shown in Table 2. Primary kidney diseases were 

shown in Table 3. A total of 37 patients (19.78%) 

had diabetes. Out of that number 22 were males 

Table 2. Distribution of patients by hemodialysis length. 

Hemodialysis length (years) No of patients (%) 

>1 41 (21.92%) 

2-5 85 (45.45 %) 

6-10 36 (19.26%) 

11-15 19 (10.16%) 

>16 6 (3.20%) 

Total 187 (100.00%) 

159

160 


Table 3. Distribution of patients by primary kidney disease 

Primary kidney disease No (%) 

Pyelonephritis 56 (30.0%) 

Glomerulonephritis 42 (22.5%) 

Nephropathia diabetica 37 (19.8%) 

Nephroangiosclerosis 17 (9.1%) 

Polycistosis renum 10 (5.3%) 

Other 25 (13.4%) 

Total 187 (100.1%) 

and 15 females. Mean age of the patients with 

diabetes was 61 years and mean hemodialysis 

length was 2 years. The research showed that 

165 patients (88.23%) had some form of CVD. 

From that number 71 were females and 94 males. 

Mean age of patients diagnosed for CVD was 

58 years and their average hemodialysis duration 

was 4,89 years (SD 2.55). Pyelonephritis was 

the most frequent kidney disease accounting for 

29.94% of patients, glomerulonephritis was the 

second (22.45%) and diabetic nephropathy the 

third (19.79%) (Table 3). 

From the total number of patients, 110 patients 

(58.82%) had hypertension, 20 patients had myocardial 

infarction, 31 patients had angina pectoris, 

24 patients had myocardiopathia and 70 patients 

had atherosclerotic aortal changes (Table 4). 

From 20 patients who have had myocardial infarction, 

9 patients (45%) had diabetes and from 

31 patients with angina pectoris, 12 (38.7%) had 

diabetes (Table 4). From the Graph 1 we can see 

that from 110 patients with hypertension, most of 

the patients (54.54%) use ACE inhibitors monotherapy. 

Most of the patients (90.0%) who have 

had myocardial infarction use a combination of 

drugs (ACE inhibitors, beta blockers, acetylsalicylic 

acid and antilipemics). Also, most of the 

patients with angina pectoris (70,97%) use combination 

of nitrates, beta blockers and acetylsalicylic 

acid (Figure 1) 

Our research showed that out of 165 patients 

with CVD, 103 patients (62.42%) had pathological 

ECG findings, with most of the patients being 

males (62). 

Table 4. Patients distribution by cardiovascular diseases 

CVD No (%) 

Mean age 

(years) 

Diabetes 

Hypertension 110 (58.82%) 57 

Myocardial infarction 20 (10.7%) 60 9 (45.0%) 

Angina pectoris 31 (16.58) 61 12 (38.7%) 

Myocardiopathia 24 (12.84%) 69.5 

Atherosclerotic changes 

on aortal valve 

70 (37.44%) 60 

Table 5. Laboratory parameters in non diabetics* 

Parameter Mean Minimum Maximum SD 

Hemoglobin 114,4 71,9 165 15,6 

Calcium 2,23 1,67 2,94 0,21 

Phosphorus 1,81 0,74 3,14 0,46 

Parathormone 457,15 3,4 1673 346 

Cholesterol 4,44 1,82 9,4 1,23 

Trigliceride 1,82 0,38 8,2 0,98 

CRP 8,04 0,2 80 12,36 

Uric acid 397,22 39,6 572 70,19 

Albumin 36,79 21 46 3,65 

*CRP, C Reactive Protein; SD, standard deviation 

Laboratory analyses have been performed for 

all patients. Results of these examinations were 

shown in Table 5 and Table 6. Using Student ttest 

we showed statistically significant difference 

between parathormone in non diabetics (mean 

457.15, SD 364), and diabetics (mean 287,88, 

SD 257.45) at the level p=0,021. Calculations of 

other laboratory parameters showed no statistical 

significance at the level p

Figure 1. Anti-hypertensive therapy in the group of patients 

with hypertension 

ble 2). Average hemodialysis length of the study 

population was 4 years. Analyzing Student t-test 

results we can see that there is no statistical significance 

between the age in patients with and 

without CVD (p< 0.05). Patients with CVD have 

been twice as long on hemodialysis compared to 

patients without CVD, with statistical significance 

at the level p

162 


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5. 

6. 

US Renal Data System. USRDS 2004 Annual Data 

Report: Atlas of end-stage renal disease in the United 

States, Bethesda. National Institutes of Health, 

National Institute of Diabetes and Digestive and 

Kidney Diseases, 2004. 

U.S. Renal Data System: USRDS 2002 Annual Data 

Report, in, Bethesda, MD. National Institutes of 

Health, National Institute of Diabetes and Digestive 

Diseases, 2002. 

US Renal Data System: USRDS 2004 Annual Data 

Report: Atlas of End-Stage Renal Disease in the 

United States, Bethesda, National Institutes of Health, 

National Institute of Diabetes and Digestive and 

Kidney Diseases, 2004. 

7. Mimura I., Nishi H., Mise N., Mori M. and Sugimoto 

T. Left ventricular geometry and cardiovascular 

mortality based on haemodialysis patient autopsy 

analyses. Nephrology 2010; 15:549–54. 

8. Levin A, Singer J, Thompson CR, Ross H, Lewis M. 

Prevalent left ventricular hypertrophy in the predialysis 

population: identifying opportunities for intervention. 

Am J Kidney Dis 1996; 27:347-54. 

9. Schönenberger A, Winkelspecht B, Köhler H, Girndt 

M. High prevalence of aortic valve alterations in 

haemodialysis patients is associated with signs of 

chronic inflammation. Nephron Clin Pract 2004; 

96:c48-c55. 

10. Lameire N. Cardiovascular problems in ESRD patients. 

Nefrología 2000; 20 (Supl 3):37. 

Analiza dijagnostike i liječenja kod kardiovaskularnog komorbiditeta dijaliznih pacijenata 

– iskustvo jednog centra 

Halima Resić 1 , Besim Prnjavorac 2 , Fahrudin Mašnić 1 , Selma Ajanović1 , Nihad Kukavica1 , Amela 

Bećiragić1 1 2 Klinika za hemodijalizu, Klinički centar Univerziteta u Sarajevu, Sarajevo; Centar za hemodijalizu, Opća bolnica Tešanj, Tešanj; Bosna i 

Hercegovina 

SAŽETAK 

Cilj Analizirati učestalost kardiovaskularnih bolesti u dijaliznoj populaciji, te analizirati najčešće dijagnostičke 

i terapijske postupke. 

Metode U studiji je analizirano 187 dijaliznih bolesnika, 106 muškaraca i 81 žena, podijeljenih u 

skupine dijabetičara i nedijabetičara. Analiza je uključivala anamnezu, EKG, rtg pluća i srca, ehokardiogram, 

plazmatske koncentracije kalcija (Ca), fosfora (P), parathormona (PTH), holesterola (Chol), 

triglicerida (TG), C-reaktivnog proteina (CRP), hemoglobina (Hb) i mokraćne kiseline, te najčešće 

korištenu terapiju. 

Rezultati Prosječna starost pacijenata bila je 58,0 godina, najviše između 51 i 60 godina. Prosječno 

trajanje hemodijalize bilo je 4 godine, a najčešće primarne bolesti bubrega bile su pijelonefritis i glomerulonefritis. 

19,78% bolesnika imalo je dijabetes, a 165 pacijenata (88,23%) imalo je kardiovaskularne 

bolesti, najčešće hipertenziju (58,2%). U terapiji su najviše bili zastupljeni ACE inhibitori, sami ili uz 

beta-blokatore. 103 pacijenta (62,42%) imali su promjene na EKG-u. Koristeći test multiple korelacije 

nađena je korelacija, između ostalih, za BMI, Ca, P i mokraćnu kiselinu, albumin i PTH kod dijabetičara, 

na statističkoj razini značajnosti p


Adenosine deaminase and C-reactive protein in diagnosing and 

monitoring of rheumatoid arthritis 

Milada Nalesnik 1 , Jasminka Mehanović Nikolić 1 , Slavica Jandrić 1,2 

1 School of Medicine of the University of Banjaluka, 2 Institute for Physical Medicine and Rehabilitation “Dr Miroslav Zotović” Banjaluka; 



Jasminka Mehanović Nikolić 

School of Medicine, University of 

Banjaluka 

Save Mrkalja 14, 78000 Banja Luka, 


Phone: +387 51 234123; fax.: +387 51 

234123 

E-mail: nikolicjasminka@hotmail.com 





Accepted: 


ABSTRACT 


Aim To determine of catalytic activities of adenosine deaminase 

(ADA) and values of C-reactive protein (CRP) concentration in 

serums of patients with rheumatoid arthritis (RA), who were and 

were not treated with Methotrexate (MTX), and identifying the 

possibilities of using these biochemical parameters in diagnosing 

and monitoring of treatment effects in RA. 

Methods The study involved 120 subjects (60 healthy ones, who 

are in accordance with examined groups concerning age and sex, 

30 suffering from RA who were not treated with MTX and 30 

suffering from RA who were treated by MTX). Catalytic activities 

of ADA in serum were determined by spectrophotometric method 

using adenosine as a substrate. CRP concentrations in serum were 

determined immunoturbidimetrically. 

Results A statistically significant correlation between values of 

ADA catalytic activities and values of CRP concentrations (r=0.55, 

p0.01). 

Conclusion Study results have shown that ADA catalytic activity 

in serum can be a useful biochemical marker of inflammatory 

process in RA. 

Key words: adenosine deaminase, C-reactive protein, rheumatoid 

arthritis, methotrexate 

163

164 


INTRODUCTION 

Rheumatoid arthritis (RA) is an inflammatory system 

autoimmune disease characterized by chronic 

proliferative synovitis that leads to articular 

cartilage and subchondral bone destruction (1), 

less attacking other organs with consequential disability 

development, working ability decreasing 

and premature death (2). The prevalence of RA, 

the most common system disease of connective 

tissue, is 1-2% of the world population (3). Etiology 

of RA, as well as etiology of most rheumatic 

diseases, is still to a great extent unknown and 

unclear. Primary pathological process in RA is 

happening in the area of synovial membrane of 

a joint which thickens a lot and becomes edematous 

because of the inflammation (synovitis), and 

it is infiltrated with mononuclear cells, together 

with growing in of the new blood vessels (4). 

A problem in diagnosing occurs because of the 

most often present typical and gradual start of RA 

with unclear and uncharacteristic clinic signs and 

asymptomatic intervals (2). 

In clinical practice, RA is diagnosed respecting 

revised criteria of ACR (American College of 

Rheumatology) from 1987 and according to them 

four positive criteria are needed for RA diagnosing, 

having in mind that the first four criteria 

must last at least for 6 weeks (2). ACR criteria 

for RA are mostly used as a “golden standard” 

in RA diagnosing although they have a limit for 

early diagnosing of RA, having in mind that they 

mainly comprise manifest clinical symptoms 

which are not necessarily shown in early stage 

of the disease (2). At the same time, those are the 

reasons why it is necessary to find a new “golden 

standard” that would be less dependent on RA 

clinical symptoms (2). 

Although there is still no safe and specific laboratory 

test, laboratory diagnosing is very important 

in diagnosing RA and presents a parameter for 

assessment of inflammatory process activity and 

monitoring of treatment effects (5). 

C-reactive protein (CRP) presents an indicator of 

RA progress (6). It is present in active phase of RA 

in almost all the subjects, and it is in a good correlation 

with erythrocyte sedimentation speed (6). 

Adenosine deaminase (ADA, adenosine amino 

hydrolase, E.C.3.5.4.4) presents an enzyme which 

catalyses irreversible hydrolytic deamination 

of adenosine to inosine and 2`deoxyadenosine to 

2`deoxyinosine together with ammonium isolation 

(7). ADA has a metabolic significance for its 

part in catabolism of purine derivates of adenosine 

and deoxyadenosine, and it is also significant 

for proliferative activity of immunocompetent 

and hematopoietic cells (7). 

This enzyme congenital deficiency leads to serious 

immune-deficiencies in which defect in 

metabolism of B lymphocytes leads to hypo- and 

a-gammaglobulinemia, as T lymphocytes are not 

able to take part in immunological reactions (7). 

In congenital deficiencies of ADA, appearance 

of lymphopenia is also a result of toxic effects 

of increased concentration of adenosine and deoxyadenosine 

due to ADA insufficiency (8,9) and 

on the other hand increased concentration of nucleotides 

and their derivates leads to inhibition of 

ribonucleotide reductase and consequentially to 

inhibition of DNA synthesis (10). 

Medical treatment of RA is characterized with 

complexity and multidisciplinarity, but thanks to 

better understanding of pathophysiological mechanisms 

of the disease there was a change in therapeutic 

treatment of RA (2). Today a remodeled 

therapy pyramid, which comprises usage of drugs 

that modify the disease (DMARDs - Disease 

Modifying Anti Rheumatic Drugs), is used (2). 

DMARDs present basic drugs in therapy of the 

system diseases of connective tissue, and there is 

also Methotrexate (MTX), which for its therapy 

efficiency and toxicity profile now presents one 

of the possible drugs in RA therapy, and monotherapy, 

and more often in combination with other 

drugs (2). For technical reasons (half-life of adenosine 

in blood lasts for about two seconds) it is 

very difficult to show experimentally the influence 

of MTX on adenosine metabolism in RA (11). 

As purine metabolism has a significant role in RA 

(12), and anti-inflammatory effect of MTX is mediated 

by stimulation of adenosine receptors (13), 

there is an assumption that certain answers regarding 

metabolism and therapy effects of MTX could 

be provided by catalytic activity of adenosine 

deaminase in serum. It is proved that MTX inhibits 

ADA, decreases V ADA in lymphocytes 

max 

and increases vasodilatation induced by adenosine 

(14). By in vitro experiments, it is proved that 

MTX inhibits activity of vascular endothelia and 

mononuclear cells in peripheral blood, decreases

production of RF, lowers the level of IL-6, IL-8, 

and receptors for TNF-α, and IL-2 (15). 

All the aforementioned mechanisms of MTX influence 

on adenosine metabolism and ADA catalytic 

activity point out the significance of determination 

of ADA catalytic activity in serum in RA 

as a possible biochemical parameter for monitoring 

the therapy effects in diseased subjects who 

were treated with MTX (16-19). 


Patients 

The study involved 120 subjects who were divided 

into three groups. In the first group were the 

patients suffering from rheumatoid arthritis hospitalized 

in Health Center ‘Spa Vrucica’ in Teslic, 

the Institute for Physical Medicine and Rehabilitation 

“Dr Miroslav Zotovic” in Banjaluka, or 

they were treated in a dispensary of family medicine 

in the Health Center in Banjaluka. The control 

group consisted of 60 healthy subjects (46 

women and 14 men) with average age of 52.78 

years (52.78 ± 3.95) and they were from 28 to 74 

years old with no family members suffering from 

rheumatoid arthritis and medical treatment. Out 

of the remaining 60 subjects (group A and group 

B), 20 were suspected to suffer from rheumatoid 

arthritis and 40 were diagnosed with rheumatoid 

arthritis. All the subjects were diagnosed with RA 

by subspecialists rheumatologists. Criteria for involving 

the patients in the study and their exclusion 

were the revised ACR criteria from 1987. The 

group A consisted of 30 subjects in acute stage of 

RA (25 women and 5 men) with average age of 

56.67 years (56.67 ± 8.54) and they were from 

41 to 74 years old. Twelve subjects were suspected 

to suffer from RA in the period between three 

and six months (they were not treated with MTX 

within the medical therapy for RA), and the diagnoses 

for RA were confirmed during our study. 

Ten subjects were the new ones suffering from 

RA who were not treated by MTX within the medical 

therapy for RA. The group B consisted of 

30 subjects (27 women and 3 men) with average 

age of 56 years (56 ± 13.54), and they were from 

29 to 74 years old. Within this group, all the subjects 

were treated with common therapy dose of 

MTX within the medical therapy for RA during 

the period of more than two months. 

Nalesnik et al ADA and CRP and rheumatoid arthritis 

Methods 

Catalytic activity of ADA was determined by 

Giusti`s modified spectrophotometric method 

(20) using adenosine (Sigma, Aldrich) as a substrate, 

and the results were read by spectrophotometer 

Spekol 11. 

The method principle was the following: by adenosine 

deaminase reacting, ammonium is released 

from adenosine and together with phenolnitroprusside 

reagent and sodium hypochlorite 

alkaline solution gives indophenol blue colour 

whose intensity is proportional to the amount of 

released ammonium, that is catalytic activity of 

total adenosine deaminase. 

The following reagents were used: phosphate buffer 

(50 mM, pH 6.5) (reagent I), buffered adenosine 

solution (21 mM adenosine, 50 mM phosphate, 

pH 6.5) (reagent II), phenol-nitroprusside 

solution (106 mM phenol; 0.17 mM nitroprusside) 

(reagent III), alkaline hypochlorite solution 

(11 mM NaOCl; 125 mM NaOH) (reagent IV), 

ammonium sulphate stock solution (15 mM) (reagent 

V), ammonium sulphate standard solution 

(75µM, NH /mL) (reagent VI). 

3 

All reagents are kept at the temperature of 0° to 

4°C. Reagents I, III, V (in dark bottles) and IV 

were stable for more than two months. Adenosine 

crystallizes at the temperature of 4°C. ADA is 

stable in serum samples for 24 hours at 25°C, but 

at 4°C it is stable for at least seven days (21, 22). 

Procedure of ADA catalytic activity in serum determination 

is shown in Table 1. Referent interval 

of ADA catalytic activities for this method was 

13.20-20.80 U/L (22). 

CRP concentrations in serum were determined 

immunoturbidimetrically (Roche Diagnostic, 

Mannheim, Germany) (Hitachi 904 analyzer). 

According to producers’ recommendation, the limit 

for decision making was CRP concentration 

value 8 mg/L. 


Study results are expressed in a form of arithmetic 

mean ± standard deviation ( X ± SD 

). Testing 

the significance of differences between the control 

group and group A (subjects who did not get 

medical therapy) and group B (subjects who had 

medical therapy), was done by the standard Student 

t-test based on results of distribution. Valu- 

165

166 


es p

increased ADA catalytic activity is registered in 

relation to healthy subjects, and decreased ADA 

catalytic activity in serum was registered in subjects 

with RA who were treated with MTX in 

relation to healthy subjects. The results were in 

accordance with described mechanism of MTX 

action, by adenosine metabolism, actually with 

ADA inhibition (11-15). 

In our study we determined a statistically significant 

correlation between ADA catalytic activities 

and CRP concentrations in serums of subjects in 

the group A (r=0.55, p0.01). 

According to our results we can conclude that 

CRP is not a good indicator for monitoring of 

therapy effects in RA whereas the ADA catalytic 

activity in serum has shown a good diagnostic 

importance as a biochemical marker of an inflammatory 

process in RA. 



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adenosine and blood flow. Ann Rheum Dis 2006; 

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Ann Rheum Dis 2002; 61:1060-4. 

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the treatment of rheumatoid arthritis. Pharmacol Rev 

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Adenozin deaminaza i C-reaktivni protein u dijagnostici i praćenju reumatoidnog artritisa 

Milada Nalesnik1 , Jasminka Mehanović Nikolić1 , Slavica Jandrić1,2 1 2 Medicinski fakultet Univerziteta u Banjaluci, Institut za fizikalnu medicinu i rehabilitaciju „Dr. Miroslav Zotović“, Banjaluka; Bosna i 

Hercegovina 

SAŽETAK 

Cilj Odrediti katalitičke aktivnosti adenozin deaminaze (ADA) i vrijednosti koncentracija C-reaktivnog 

proteina (CRP) u serumima oboljelih od reumatoidnog artritisa (RA), koji su bili sa ili bez terapije Metotreksatom 

(MTX), kao i utvrđivanje mogućnosti uvođenja ovih biohemijskih parametara u dijagnostiku 

i praćenje terapijskih efekata u RA. 

Metode Istraživanjem je obuhvaćeno 120 ispitanika (60 zdravih ispitanika koji prema starosti i polu 

odgovaraju ispitivanim grupama; te 30 bolesnika s RA, bez terapije MTX, i 30 bolesnika s RA koji su 

dobijali terapiju MTX). Katalitičke aktivnosti ADA u serumu su određivane spektrofotometrijskom metodom, 

uz adenozin, kao supstrat. Koncentracije CRP u serumu određivane su imunoturbidimetrijski. 

Rezultati Utvrđena je statistički značajna korelacija između vrijednosti katalitičkih aktivnosti ADA i 

vrijednosti koncentracija CRP (r=0,55, p0,01). 

Zaključak Rezultati istraživanja su ukazali da određivanje katalitičke aktivnosti ADA u serumu može 

biti koristan biohemijski marker upalnog procesa kod oboljelih od reumatoidnog artritisa. 

Ključne riječi: adenozin deaminaza, C-reaktivni protein, reumatoidni artritis, metotreksat. 

Original submission: 02 November 2010; Revised submission: 13 December 2010; Accepted: 14 December 2010.

MEDICINSKI GLASNIK - Aktuelno Ljekarska komora ZE - DO kantona

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